After exposure to mucosal ribotoxic stress, gut epithelial cells use defensive transporters to excrete the toxins in the feces. The most well known is the ATP-binding cassette (ABC) transporter, which is specialized for exporting toxic xenobiotics. Intestinal epithelial cells also use some ABC transporters, such as the multidrug resistance-associated protein 1 (MDR1, ABCB1) and MRP2 (ABCC2), to export ribotoxic trichothecenes [31,32]. MDR1 is expressed highly in the intestinal epithelia, renal tubular epithelia, canalicular membrane of hepatocytes, cerebral capillary endothelial cells, testes, and ovaries. MDR1 exports a broad range of hydrophobic compounds, and thus functions as a biological barrier by preventing the exposure of tissues to toxic substances. MDR1 is also a secretory transporter that limits the entry of toxic xenobiotics into the gut epithelial cells. In contrast to the defensive roles played by ABC transporters, retroviral induction of human MDR1 cDNA, which encodes ABCB1, triggers major accumulation of globotriaosylceramide, a glycosphingolipid receptor for the ribotoxic shiga toxin, leading to a million-fold increase in barrier cell sensitivity to toxins [33]. The human MDR1 gene is located on chromosome 7 (7q21.1), a susceptibility locus for inflammatory bowel disease (IBD) [34,35]. A meta-analysis study demonstrated that a significant number of IBD patients harbor mutations in this gene and develop a severe spontaneous colitis that is characterized by impaired functionality of the intestinal barrier and immune reactivity to luminal xenobiotics, such as ribotoxic agents. Moreover, ribotoxic xenobiotics modulate nutrient transport by interfering with the d-glucose/d-galactose sodium-dependent transporter (SGLT1), the d-fructose transporter GLUT5, active/passive l-serine transporters, and the passive transporters of d-glucose (GLUT) [23,36]. The selective effects of ribtoxic stress on intestinal nutrient transport may account for the malnutrition and weight loss observed in toxin-exposed animals and human. A suppressed nutrient transport system provides a potential mechanism by which ribotoxic stress could induce diarrhea in animals and humans. For instance, exposure to low amounts of ribotoxic deoxynivalenol can cause aqueous diarrhea by inhibiting the intestinal SGLT1 transporter, which results in an imbalance of epithelial cellular water due to decreased d-glucose-associated water absorption. Moreover, exposure to toxic levels of deoxynivalenol may inhibit SGLT1 function and disrupt the epithelial barrier, leading to inflammatory diarrhea.

The intestinal mucosal barrier has evolved to maintain a delicate balance between the absorption of essential nutrients and the prevention of harmful xenobiotic entry and responses. Junction structural alterations can be detected in the gut epithelial barrier in both human IBD and experimental models of intestinal inflammation. Leaky gut symptoms are linked to an increase in intestinal permeability and a decrease in transepithelial resistance. The intestinal epithelial junctions consist of desmosomes, adherent junctions, and tight junctions, all of which modulate both intercellular adhesion and paracellular transport. E-cadherin plays an important role in maintaining the integrity of the intestinal barrier; its cellular localization is disrupted in patients with Crohn’s disease (CD) [37]. However, the abnormal expression and dislocalization of tight junction structural proteins, such as hyperphosphorylated occludins, zonula occludens, and claudin family proteins, are frequently observed in IBD pathogenesis [38]. The tight junctions, located at the apical end of the intercellular space, are regulated highly by cytokines, which play critical roles in the modulation of intestinal barrier function. Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interferon γ, interleukin (IL)-1β, IL-13, and a cellular ligand for herpes virus entry mediator and lymphotoxin receptor (LIGHT), promote barrier dysfunction by inhibiting the transcription of junction proteins and inducing cytoskeleton-mediated redistribution of tight junction proteins [39,40]. For instance, some pro-inflammatory cytokines enhance the expression of myosin light-chain kinase, which leads to myosin II re-arrangement to allow interaction with tight junction proteins. Re-organized tight junction proteins are then able to exit the complex via the endocytic pathway. In contrast, regulatory cytokines, including transforming growth factor (TGF)-β, are involved in the re-constitution of tight junctional complexes [41]. The gut epithelia in IBD patients displays a leaky barrier of decreased junctional complexity, with a lower number of tight junction strands, reduced depth of the primary tight junctional meshwork, increased apoptotic rate, and appearance of strand discontinuity, all of which have been associated closely with increased uptake of luminal food and bacterial antigens [42]. Because epithelial barrier disruption increases host susceptibility to acute microbial infections and to chronic hypersensitive diseases, including IBD, various ribotoxic stresses have been studied extensively for their effects on the tight junctions of the epithelial barrier [43,44,45,46,47,48,49]. In response to ribotoxic deoxynivalenol, the intestinal epithelial barrier in the pig (the most susceptible species) demonstrated injury by decreased expression of the tight junction protein, claudin-4 [47,48]. Decreased claudin-4 leads to a loss of epithelial cell integrity and disruption of epithelial polarity. A recent study demonstrated that, depending on the route of toxin exposure (direct apical versus basolateral via the circulation), the polarized gut epithelial cell layer responded differently to ribotoxic deoxynivalenol. Basolateral exposure of the gut barrier causes significantly more vulnerability to the tight junctional breakdown than exposure to the apical surface [44]. The reduced quantity of tight junction proteins is mediated by ribotoxin-activated p44/42 extracellular signal-regulated kinase (ERK) MAPK. Ribotoxic stress induces various pro-inflammatory cytokines, including TNF-α and IL-1β. These cytokines may play important roles in dysregulating the tight junctional complex and decreasing epithelial integrity, as suggested in IBD pathogenesis. The specific association between pro-inflammatory cytokines and epithelial barrier disruption is implicated in ribotoxin ricin-intoxicated animal model [26]. Taken together, ribotoxic stresses promote the leaky gut environment by disrupting junctional structures, which allows for the translocation of luminal bacteria and for the subsequent over-stimulation of underlying lymphoid systems. The ribotoxic stress may facilitate overload of luminal contents (e.g., enteric bacteria) through the leaky barrier, which may demonstrate the potent etiological factors of human IBD.

Epithelia with regions of apoptosis are susceptible to conductive leakage, which leads to barrier dysfunction, bacterial translocation and subsequent infection. Increased epithelial apoptosis is another critical factor of compromised barrier integrity in both UC and CD patients [50,51,52,53]. In early stages of mild to moderate inflammation in UC, apoptosis accounts for approximately half of the impaired colorectal conductivity whereas the other half is caused by degradation of tight junctions in non-apoptotic areas [54]. Erosion and ulcer-type lesions associated with the severe mucosal and systemic inflammatory response dominate the late stages of the disease [42]. In dextran sulfate sodium (DSS)-induced colitis, the colonic epithelia demonstrate increased apoptosis and decreased proliferation, which may lead to the breakdown of the epithelial barrier, and thus facilitate the mucosal invasion of enteric bacteria [55]. During IBD pathogenesis, some species of commensal enteric bacteria, including E. coli O4, may breach the epithelial barrier by forming apoptotic loci or focal leaks at points of bacterial penetration. Mucosal ribotoxic xenobiotics can also induce epithelial apoptosis in the gastrointestinal tract in different exposure models [18,44,45,46,47,48,49,50,51,52,53,54,55,56]. Intestinal epithelia are more resistant than lymphocytes to the cytotoxic actions associated with ribotoxic stress, but the local concentration of ribotoxins in the gastrointestinal tract following dietary ingestion is generally much higher than ribotoxin levels in the circulation and other distributed tissues. Thus, damage-mediated action of a higher intensity could be expected in the intestinal epithelium. However, intramuscular exposure to ribotoxic stress can lead to severe small intestinal injuries as well as the infiltration of large numbers of plasma cells into the lamina propria and subsequent apoptosis of mucosal lymphocytes [46,56]. Regarding signaling pathways, ribotoxic agents activate p38 MAPK and Jun N-terminal kinase (JNK), which are involved in pro-apoptotic caspase 3 activation in intestinal epithelia [18,57]. MAPK also can mediate negative regulation of apoptosis induction; e.g., extracellular signal-regulated kinases (ERK) 1/2 can directly phosphorylate caspase 9 in response to growth factor [58]. Caspase 9 phosphorylation is critical to restraining apoptosis during mitotic arrest by Bcl2 phosphorylation, whereas the proteolytic cascade of caspase 9 leads to apoptosis. The ERK1/2 signal also can protect cells from ulcerative insults by enhancing epithelial proliferation and cell survival [59]. ERK1/2 signal is activated in response to ribotoxic stress-mediated pro-apoptotic signals to mediate expression of early growth response gene 1 product (EGR1) [60], which can trigger epithelial reconstitution as well as mitotic cytokine production. Ultimately, the fate of ribotoxin-exposed epithelial cells is determined by the counteracting balance between pro-apoptotic signals and survival responses, dependant on the signaling context.

Some ribotoxic agent-induced cell deaths are independent of MAPK activation. Several trichothecenes, including acetyl T-2, T-2 toxin, and verrucarin, inhibit protein synthesis without activating JNK and lead to caspase activation, which indicates that JNK and p38 kinases are not necessary for caspase activation [57]. As another MAPK-independent pathway, ribotoxic stress can elicit endoplasmic reticulum (ER) stress. ER stress induces calcium (Ca²⁺) release in leukocytes—Ca²⁺-dependent calpain activation and subsequent pro-apoptotic caspase 8 activation [61]. Ribotoxic trichothecenes can also induce ER stress in gut epithelial cells, but it is not linked to cellular apoptosis [62]. Ribotoxin-induced epithelial ER stress mediates pro-inflammatory cytokine production instead of apoptosis [63], which may contribute to the disruption of epithelial tight junctions, as suggested in section 2.2. However, since local intestinal ribotoxic doses are generally higher than those in circulation, extremely high doses of ribotoxic agents can induce epithelial cell death. In addition to caspase-mediated cell death, ribotoxic anisomycin induces macrophage inhibitory cytokine 1 (MIC-1) gene expression, which is a critical inducer of intestinal cellular apoptosis and barrier disruption [64]. MIC-1, which is also known as PTGF-β, PLAB, GDF15, PDF, NAG-1, and PL74, is a cytokine from the transforming growth factor-β superfamily and is involved in epithelial pathogenesis [65,66,67]. Little to no detectable expression of MIC-1 is observed in normal epithelial cells. However, the level of MIC-1 expression rises dramatically with epithelial neoplastic transformation, and MIC-1 expression increases further in response to a variety of anti-tumorigenic stimuli, including gamma irradiation and chemo-preventive agents—the latter includes non-steroidal anti-inflammatory drugs (NSAIDs) and natural products [68,69]. NSAIDs induce ulcerative lesions similar to those induced by ribotoxic xenobiotics, and the pro-apoptotic action of NSAIDs in the gut epithelium is mediated by MIC-1 protein though ER stress [70]. Although the mechanism of MIC-1-mediated apoptosis is not clearly understood in the gut epithelium, several lines of evidence indicate that since the promoter of MIC-1 has a potential promoter binding site for p53 protein, MIC-1 can mediate p53-dependent growth suppression [68,71,72]. However, MIC-1 can induce apoptosis independent of the p53 pathway [73]. MIC-1 is expressed in colorectal cancer tissues and has strong associations with serum levels and malignancy in the gut mucosa [65]. Moreover, MIC-1 induces tumor metastasis by triggering the FAK-RhoA signaling pathway, which leads to actin reorganization [74]. Chronic exposure to the ribotoxic stress can maintain prolonged MIC-1 expression, which may be linked to intestinal neoplastic progression. Therefore, further investigations are needed to examine other MIC-1-mediated effects on epithelial cells chronically exposed to ribotoxic agents, whereas MIC-1 effects induced by acute exposure can be pro-apoptotic.