Impacts of trichothecene mycotoxins on human colonic epithelial cells: molecular mechanisms and signaling pathways

Shao-Ji Li; Chun-Min Yang; Shiyi Ou

doi:10.1016/j.jare.2025.11.024

. 2025 Nov 16;85:257–279. doi: 10.1016/j.jare.2025.11.024

Impacts of trichothecene mycotoxins on human colonic epithelial cells: molecular mechanisms and signaling pathways

Shao-Ji Li ¹, Chun-Min Yang ^1,^⁎, Shiyi Ou ^1,^⁎

PMCID: PMC13316413 PMID: 41253273

Graphical abstract

Keywords: Trichothecenes, Deoxynivalenol, T-2, Colonic epithelial cell, Toxic effects

Highlights

•
Offering a structured knowledge framework from observations and basic concepts of toxic effects to profound understanding.
•
In-depth elucidation of molecular mechanisms and signaling pathways underlying trichothecene toxicity in human CECs.
•
Integration of various toxic effects into a unified figure, with careful layout to illustrate their interrelationships.
•
A comprehensive structural compilation of all discussed trichothecenes and comparison in their cytotoxicity.

Abstract

Background

Trichothecenes are a family of structurally related mycotoxins typically found in cereal crops, thus threatening the health of human digestive tract. In colonic epithelial cells (CECs), trichothecenes inhibit protein synthesis and cell proliferation, induce oxidative stress, DNA damage, ribotoxic stress response (RSR), endoplasmic reticulum stress (ERS), unfolded protein response (UPR), inflammation, and apoptosis, destruct cell junctions, and alter mucus composition. Although recent studies have enhanced the understanding on the molecular mechanisms of these toxic effects, interconnections between these toxic effects were poorly established.

Aim of review

This review comprehensively summarizes the influence of trichothecenes on CECs. This review emphasizes the elucidation of molecular mechanisms and signaling pathways underlying trichothecene toxicity, in an attempt to integrate diverse toxic effects into a unified mechanistic framework.

Key scientific concepts of review

Trichothecenes inhibit protein synthesis by binding to the ribosomal A-site, thus triggering RSR to activate MAPKs via PKR or ZAKα. Trichothecenes induce ERS and UPR, mediated by PERK/eIF2/ATF4 and Ire1α/XBP1 signaling, which potentially activate CHOP, MAPKs, p53, and NF-κB. Trichothecenes disturb the mitochondrial electron transport chain to overproduce ROS, further activating NF-κB. Trichothecenes induce nitric oxide overproduction via inducible nitric oxide synthase, which is transcriptionally activated by NF-κB. Trichothecenes may cause DNA damage by direct interaction or by oxidative stress, thereby activating ATM/p53 signaling. Trichothecenes modulate inflammation via MAPK, NF-κB, JAK/STAT, and UPR pathways. The cytotoxicity of trichothecenes depends on molecular structure, exposure dose, and cell differentiation status. Trichothecenes suppress cell proliferation via Wnt and/or JAK/STAT pathways, and induce apoptosis via mitochondrial and Fas pathways by regulating p53, CHOP, and BCL-2 family proteins. Trichothecenes impair epithelial integrity by reducing cell junction proteins through MAPK, Wnt and JAK/STAT pathways, and by inhibiting tight junction assembly through inactivating PKA. Trichothecenes reduce mucin production through MAPKs and IRE1β signaling.

Introduction

Introduction of colonic epithelium

The intestinal epithelium is a cell monolayer of intestinal epithelial cells (IECs) which constantly differentiates from a stem cell in the crypt [1]. The integrity of an intact intestinal epithelium is maintained by intercellular proteinaceous cell junctions, including tight junction (TJ), adherens junction (AJ), desmosome, and gap junction (GJ). TJs serve as regulators of epithelial paracellular permeability. TJs are composed of the transmembrane proteins claudins, occludin, and junctional adhesion molecules (JAMs), and structurally connected with the intracellular cytoskeleton via zonula occludens (ZO) proteins as scaffolds. Claudins form TJ strands through homophilic and heterophilic interactions. TJ strands encircle the cells like zippers, which seal the intercellular space to restrict paracellular diffusion of molecules. The amount of TJ strands determines paracellular permeability. AJs and desmosomes (collectively known as anchoring junctions) hold the connection of adjacent epithelial cells tightly but do not seal the intercellular space. AJs encircle the cells like belts, and are formed by epithelial cadherin (E-cadherin), a calcium-dependent transmembrane adhesion protein. E-cadherin has an ectodomain and a cytoplasmic domain. The ectodomain mediates adhesion recognition and homophilic binding, while the highly conserved cytoplasmic domain connects with the intracellular cytoskeleton via p120, α-, and β-catenins as linkers. GJs mediate intercellular communication. Both desmosomes and GJs are restricted to particular sites between adjacent epithelial cells [2,3]. The intestinal epithelium is rapidly proliferating and renewing, with a complete turnover every 24–96 h. Epithelial renewal takes place through a combination of cell division, cell migration (also known as restitution) and surface shedding [4]. Different from epithelium of the small intestine, the colonic epithelium does not form villi [5], and is covered by two (inner and outer) mucus layers. The inner mucus layer is dense, and anchored to the goblet cells and attached to the epithelium, while the outer mucus layer is thick, loose, and unattached, and harbors intestinal bacteria [6]. The mucus consists of mucins, resistin-like molecule β (RELM-β), trefoil factors (TFFs), and other bioactive components produced and secreted by goblet cells, the specialized secretory IECs [7]. The mucins are glycosylated proteins expressed from up to 22 mucin genes, i.e. from MUC1 to MUC22, with MUC2 as the predominant mucin in the intestine [8]. Compared with epithelium of the small intestine, the colonic epithelium has a higher level of RELM-β[9], which functions as an immunoregulator and a regulator of MUC2 transcription and secretion [7]. TFFs are bioactive peptides which stimulate cell migration and restitution, so they are essential in epithelial repair [4]. Overall, the colonic epithelium coordinates nutrient absorption and recycling, mediates host–microorganism interactions, modulates mucosal immunity, and forms a mucus barrier [10]. However, its function is subject to impact of xenobiotic agents such as mycotoxins, which may lead to epithelial dysfunction. The fast renewal of the colonic epithelium probably makes it a sensitive target for mycotoxins which inhibit protein synthesis and impair cell viability.

Introduction of trichothecene mycotoxins

Trichothecenes are a family of structurally related mycotoxins produced by diverse filamentous fungal genera such as Fusarium, Myrothecium, Stachybotrys, Trichoderma, Trichothecium, and Spicellum, typically in agricultural products such as wheat, barley, oats, rye, and maize. Trichothecenes are non-volatile, low molecular weight (typically 200–500 Da) sesquiterpenoids, sharing a common 12,13-epoxytrichothec-9-ene (EPT) scaffold, which is a tetracyclic structure consisting of a cyclohexene ring, a tetrahydropyran ring, a cyclopentyl ring, and a C12,C13-epoxide ring [11]. Substitution can exist at one or more sites among C3, C4, C7, C8, and C15 of the EPT scaffold. The substitution groups can be hydroxyl group, carbonyl group, acetoxy group, epoxide ring, and macrocyclic ring, etc., as well as glucose, sulphate, and glutathione groups due to metabolization by plants [11,12]. Over 200 trichothecenes have been identified in nature [13], and they are classified into Types A, B, C, and D according to the substitution pattern of the EPT scaffold [11] (Table 1). Type A is characterized by the presence of a single bond at C8, and represented by T-2, HT-2, DAS, NEO, etc. Type B is characterized by a C8-carbonyl group, and represented by DON, acetyl DONs, NIV, TCN, etc. Type C is characterized by an C7,C8-epoxide group (e.g. crotocin). Type D is characterized by a C4,C15-macrocyclic ring, so is also known as macrocyclic trichothecenes (e.g. verrucarins, roridins, satratoxins) [11,13]. The biological activities of trichothecenes are most affected by the C12,C13-epoxide group, the C9=C10 double bond, and the substitution groups [14]. Ingestion of trichothecenes by humans and animals can cause disorders in the gastrointestinal tract, as well as in the immune, nervous, circulatory, and reproductive systems [11]. Oral exposure to trichothecenes has been found to induce epithelial lesions in the colon of animals, such as lymphocytic and neutrophilic infiltration, cubic and flattened enterocytes, necrotic debris, and increase of epithelial cell area [15,16]. Outbreak of alimentary toxic aleukia (ATA) in association with the consumption of Fusarium-infected cereals was initially reported in Russia in 1910s and later in 1940s. Retrospective studies suggested that T-2, HT-2, and related trichothecenes may have contributed etiologically to this disease [17]. Early in 1970s, trichothecenes have been found to inhibit protein synthesis as a primary toxic effect [18]. They also disrupt metabolism by downregulating nucleotide synthesis (adenosine, thymidine), altering energy metabolism (tricarboxylic acid cycle activation, carbohydrate dysregulation), and perturbing amino acid/lipid metabolism [19]. Detoxification strategies of trichothecenes can be classified into biosorption, biotransformation, and biotherapy, based on detoxification mechanisms. Biosorption involves binding toxins by biosorbents including aluminosilicate-based clays (e.g. montmorillonites) and bacterial/fungal cell wall components (e.g. mannan) to reduce the bioavailability and accelerate excretion. Biotransformation involves conversion of toxins into low toxicity or non-toxic forms by microorganisms or enzymes. For example, Eubacterium BBSH 797 is able to de-epoxidize trichothecenes. Biotherapy is an indirect strategy, which mainly uses bioactive agents (e.g. the antioxidant polyphenols) to alleviate and repair the harmful effects of toxins [20,21].

Table 1.

Molecular structures of selected trichothecenes.

Type	Trichothecene	Substitution/Insertion groups on the EPT scaffold
Type	Trichothecene	R1 (C3)	R2 (C4)	R3 (C15)	R4 (C7)	R5 (C8)	R6 (in macrocyclic ring)
A	T-2 toxin (T-2)	–OH	−O-acetyl	−O-acetyl	−H	–OC(=O)CH₂CH(CH₃)₂	/
	HT-2 toxin (HT-2)	–OH	–OH	−O-acetyl	−H	–OC(=O)CH₂CH(CH₃)₂	/
	15-deacetyl-T2	–OH	−O-acetyl	–OH	−H	–OC(=O)CH₂CH(CH₃)₂	/
	T-2-triol	–OH	–OH	–OH	−H	–OC(=O)CH₂CH(CH₃)₂	/
	T-2-tetraol	–OH	−OH	−OH	−H	–OH	/
	19-hydroxyl-T2	–OH	−O-acetyl	−O-acetyl	−H	–OC(=O)CH₂C(OH)(CH₃)₂	/
	20-hydroxyl-T2	–OH	−O-acetyl	−O-acetyl	−H	–OC(=O)CH₂CH(CH₃)CH₂OH	/
	Diacetoxyscirpenol (DAS)	–OH	−O-acetyl	−O-acetyl	−H	−H	/
	Neosolaniol (NEO)	–OH	−O-acetyl	−O-acetyl	−H	–OH	/
	Trichodermin	−H	−O-acetyl	−H	−H	−H	/
	NT-1 toxin (NT-1)	–OH	−O-acetyl	–OH	−H	−O-acetyl	/
	T2-3-glucuronide	−O-glucuronosyl	−O-acetyl	−O-acetyl	−H	–OC(=O)CH₂CH(CH₃)₂	/
	T2-3-glucoside	−O-glucopyranosyl	−O-acetyl	−O-acetyl	−H	–OC(=O)CH₂CH(CH₃)₂	/
	NX-2	−O-acetyl	−H	–OH	–OH	−H	/
	NX-3	–OH	−H	–OH	–OH	−H	/
	NX-4	–OH	−H	−O-acetyl	–OH	−H	/
	12-deoxytrichodermin	Trichodermin with C12=C13 ene group instead of the epoxy ring					/
	NX2-M1	The C12,C13-epoxide ring of NX-2 (for NX2-M1), NX-3 (for NX3-M1), or NX-4 (for NX4-M1) is opened, and the C9=C10 double bond becomes a single bond. C9 and C12 are hydroxylated, and C10 forms a covalent bond with C13.
	NX3-M1
	NX4-M1
B	Deoxynivalenol (DON)	–OH	−H	–OH	–OH	=O	/
	3-epi-DON	–OH	−H	(S)–OH	–OH	=O	/
	3-acetyl-DON (3ADON)	−O-acetyl	−H	–OH	–OH	=O	/
	15-acetyl-DON (15ADON)	–OH	−H	−O-acetyl	–OH	=O	/
	Fusarenon-X (FusX)	–OH	−O-acetyl	–OH	–OH	=O	/
	Nivalenol (NIV)	–OH	–OH	–OH	–OH	=O	/
	Trichothecin (TCN)	−H	–OC(=O)CH=CHCH₃	−H	−H	=O	/
	DON-3-glucoside (DON-3-Glu)	−O-glucopyranosyl	−H	–OH	–OH	=O	/
	DON-3-glucuronide	−O-glucuronosyl	−H	–OH	–OH	=O	/
	DON-15-glucuronide	–OH	−H	−O-glucuronosyl	–OH	=O	/
	DON-3-sulfate (DON-3-Sulf)	−SO₄^-	−H	–OH	–OH	=O	/
	DON-15-sulfate (DON-15-Sulf)	–OH	−H	−SO₄^-	–OH	=O	/
	Deepoxy-DON (DOM-1)	DON with C12=C13 ene group instead of the epoxy ring
C	Crotocin	−H	–OC(=O)CH=CHCH₃	−H	C7,C8-epoxide ring		/
C	Crotocol	−H	–OH	−H	C7,C8-epoxide ring		/

D	Verrucarin A (VA)	−H	C4,C15-macrocyclic ring		−H	−H	–CH(OH)CH(CH₃)CH₂CH₂OC(=O)–
	Verrucarin J (VJ)	−H	C4,C15-macrocyclic ring		−H	−H	–CH=C(CH₃)CH₂CH₂OC(=O)–
	Verrucarin L (VL)	−H	C4,C15-macrocyclic ring		−H	–OH	–CH=C(CH₃)CH₂CH₂OC(=O)–
	Mytoxin F	−H	C4,C15-macrocyclic ring		−H	−H	–CH=C(CH₃)CH(OH)CH₂OC(=O)–
	Mytoxin G	−O-glucopyranosyl	C4,C15-macrocyclic ring		−H	−H	–CH=C(CH₃)CH₂CH₂OC(=O)–
	Roridin J (RJ)	−H	C4,C15-macrocyclic ring		−H	−H	–CH=C(CH₃)CH(OH)(C₄H₆O₂)–
	Mytoxin D	–OH	C4,C15-macrocyclic ring		−H	−H	–CH=C(CH₃)CH(OH)(C₄H₆O₂)–
	Mytoxin E	−H	C4,C15-macrocyclic ring		−H	−O-acetyl	–CH=C(CH₃)CH(OH)(C₄H₆O₂)–
	Roridin H (RH)	−H	C4,C15-macrocyclic ring		−H	−H	–CH=C(CH₃)CH₂(C₄H₆O₂)–
	8α-hydroxyroridin H	−H	C4,C15-macrocyclic ring		−H	–OH	–CH=C(CH₃)CH₂(C₄H₆O₂)–
	8α-acetoxyroridin H	−H	C4,C15-macrocyclic ring		−H	−O-acetyl	–CH=C(CH₃)CH₂(C₄H₆O₂)–
	Epiisororidin E	−H	C4,C15-macrocyclic ring		−H	−H	–CH=C(CH₃)CH₂CH₂OCH(C₂H₅O)–
	Satratoxin G	−H	C4,C15-macrocyclic ring		−H	−H	−C₈H₁₂O₄–
	Satratoxin H	−H	C4,C15-macrocyclic ring		−H	−H	−C₈H₁₂O₃–
	12-deoxyroridin J	RJ with C12=C13 ene group instead of the epoxy ring
	12-deoxyepiisororidin E	Epiisororidin E with C12=C13 ene group instead of the epoxy ring

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Outline and aim of this review

This review summarizes the latest knowledge on the impact of trichothecenes on human colonic epithelial cells (CECs), in terms of (1) Inhibition of protein synthesis and initiation of ribotoxic stress response (RSR), (2) induction of endoplasmic reticulum stress (ERS) and unfolded protein response (UPR), (3) induction of oxidative stress, (4) induction of DNA damage and initiation of DNA damage response (DDR), (5) modulation of inflammatory response, (6) regulation of cell viability, (7) destruction of epithelial integrity, and (8) regulation of mucus composition. Special emphasis is placed on exploration and elucidation of possible mechanisms of these impacts on cellular and molecular levels. The majority of the collected knowledge comes from the studies on DON, the most widespread and best studied trichothecene, supplemented with the knowledge on T-2, NX-3, DAS, acetyl DONs, DON-3-Glu, DON-sulfates, TCN, verrucarins and others. This review is expected to help understanding on the toxicological effects of trichothecene mycotoxins on human CECs and especially on the underlying mechanisms, and provide valuable reference information for further study.

Inhibition of protein synthesis and initiation of ribotoxic stress response

Inhibition of protein synthesis

Ribosome, the organelle responsible for protein synthesis, is the primary target of trichothecenes, so inhibition of protein synthesis is the primary toxic effect of trichothecenes. Inhibition of protein synthesis will cause a series of toxic effects ranging from energy metabolism to cell death [3]. Both acute and chronic trichothecene exposures have been reported to inhibit protein synthesis in CECs. In human colonic cell lines Caco-2 [[22], [23], [24], [25]], HT-29, and HCEC-1CT [26,27], protein synthesis was dose-dependently inhibited by DON treatment. NX-3 exhibited similar inhibitory effect with DON on protein synthesis in HT-29 and HCEC-1CT cells, but its de-epoxidized derivative NX3-M1 exhibited no or tiny inhibitory effect on protein synthesis [28]. Dose-dependent inhibition of protein synthesis by TCN was also observed in HT-29 and HCT116 cells [29]. In rodent intestinal epithelial cells, inhibitory effect of different trichothecenes on protein synthesis ranks as T-2 > DAS > FusX > NIV > NX-3 > DON [30]. A trichothecene can block protein synthesis at the stages of initiation, elongation and/or termination, depending on its substitution pattern of the trichothecene molecule [31]. For example, little or no substituent at C4 makes the trichothecene a termination inhibitor; an ester group at C4 only makes the trichothecene an elongation inhibitor; an additional ester group on the C15 may inhibit initiation besides elongation; if C3 or C4 is acetylated besides esterification of C15, the trichothecene acts as an initiation inhibitor [32]. Since trichothecenes generally inhibit protein synthesis, the constantly renewing intestinal epithelium could be particularly vulnerable to trichothecenes.

Trichothecenes inhibit the function of ribosome by steric hindrance (Fig. 1A). The human ribosome (80S) has a molecular weight of 4.3 MDa: the 60S large subunit consists of 28S, 5S and 5.8S rRNAs and 47 proteins, while the 40S small subunit possesses a single 18S rRNA chain and 33 proteins [33]. Each ribosome has three positioning sites for tRNA molecules: the P-site for peptidyl-tRNA, the A-site for aminoacyl-tRNA, and the E-site for deacylated tRNA ready to exit. For elongation of a nascent peptide chain, a peptidyl bond between the peptide at P-site and the amino acid at A-site is formed by the peptidyl transferase activity of the 60S subunit [34]. The eukaryotic ribosome is structurally and functionally conserved, and the knowledge on trichothecene-eukaryotic ribosome binding mechanism is mostly from studies on yeast model by in silico modelling and X-ray crystallography. An extensive in silico screening suggested that the ribosomal A-site favorably interacts with DON, T-2, NX-3, VA, 15ADON, NEO, NIV, FusX, DAS, NT-1, HT-2, 19-hydroxyl-T2, 20-hydroxyl-T2, T-2-triol, T-2-tetraol, 15-deacetyl-T2, and DON-15-glucuronide, but not with 3-epi-DON, 3ADON, DON-15-Sulf, DON-3-Sulf, DON-3-Glu, DOM-1, NX-2, T2-3-glucuronide, T2-3-α-glucoside, T2-3-β-glucoside, or DON-3-glucuronide [35]. T-2, DON, and VA, as a representative of Types A, B, and D trichothecenes, share a similar binding mode within a binding pocket in the A-site of the 60S subunit, where a T-2, DON, or VA molecule contacts rRNA residues and induces a structural rearrangement of the peptidyl transferase center, thus hindering aminoacyl-tRNA positioning as well as formation of new peptidyl bond [34]. Further study revealed that T-2, DON, and VA molecules fit in the binding pocket formed by 25S rRNA (homologue of human 28S rRNA) by multiple interactions with the 25S rRNA. Specifically, the key toxic elements of the trichothecene molecule such as C12,C13-epoxide ring, C9=C10 double bond, and C3-OH interact intimately with the 25S rRNA at multiple sites by hydrogen bond, hydrophobic interaction, and Mg²⁺ ion coordination, etc. [36]. Any modification on the EPT scaffold can potentially make significant influence on these interactions. For instances, due to the absence of C12,C13-epoxide ring or the isomeric change, DOM-1 and 3-epi-DON form one less hydrogen bonds with the A-site of the peptidyl transferase center than DON does [37]. DOM-1 interacts poorly with the ribosome because its hydrophobic C12=C13 ene group, which replaces the C12,C13-epoxide ring of DON, is arranged improperly within a hydrophilic environment. The poor interaction of DON-15-Sulf with the ribosome is caused by improper positioning of its hydrophilic sulfate group within a hydrophobic environment. Voluminous substitution at C3 of the EPT scaffold (e.g. NX-2, 3ADON, DON-3-Glu, and DON-3-Sulf) are too close to the metal ion at the boundary of the binding site so that binding is hindered [35]. In contrast, substitutions at C4, C7, C8, C15, and C16 of the EPT scaffold (e.g. the C4,C15-macrocyclic ring of VA) face into an open cleft, which allows additional binding without steric hindrance, leading to increase in ribosomal infinity and toxicity [36]. The above-mentioned knowledge on the trichothecene-eukaryotic ribosome binding mechanism is based on the studies on yeast model, and can be appropriately extrapolated to human, yet experimental confirmation in human CECs is necessary in future study.

Although trichothecenes inhibit protein synthesis primarily by steric hindrance in ribosome, some alternative mechanisms may also be involved, including: (1) Cleavage of rRNA. DON was also found to indirectly promote cleavage of 28S rRNA at the sites A3560 and A4045 of the peptidyl transferase region by increasing the expression of endogenous RNases, thus disturbing the ribosomal function of protein synthesis [38]. (2) Alternative splicing of pre-mRNA. Low dosage of DON dramatically increased the aberrant splicing of pre-mRNA, especially on 3′ splice sites in Caco-2 cells, probably by downregulating the expression of two splicing factors, SF1 and U2AF1, which are essential for 3′ splice site recognitions [39]. The aberrantly spliced mRNA probably lowers translation efficiency. (3) Phosphorylation of eukaryotic initiation factor 2 (eIF2) (Fig. 1B). Phosphorylation of eIF2 on its α subunit (eIF2α) by protein kinase R (PKR) (See the subsection on RSR) and/or PKR-like endoplasmic reticulum kinase (PERK) (See the section on ERS and UPR) is known to inhibit mRNA translation. Accumulation of untranslating mRNA may induce formation of stress granules (SGs), which are membrane-less organelles that assemble under cellular stresses. Besides the untranslating mRNA, SGs also incorporate various RNA-binding proteins (RBPs) such as G3BP1/2, translation initiation factors, and non-RBPs such as signaling proteins. The biological functions of SGs remain unclear, but they are generally believed to function as sites of temporary mRNA storage and triage until the stress is relieved. How trichothecenes influence formation of SGs in human CECs have not been documented, making it an interesting topic for further study [40,41].

Initiation of ribotoxic stress response

Besides inhibition of protein synthesis, interaction of trichothecenes with the ribosome also launches ribotoxic stress response (RSR), characterized by the activation of mitogen-activated protein kinases (MAPKs) [42]. Three canonical MAPK pathways exist, i.e. ERK1/2, p38, and JNK MAPK pathways, and each one is a phosphorylation activation cascade relayed sequentially by MAP3Ks, MAP2Ks, and MAPKs [31]. Some trichothecenes are potent ribotoxic stressors. Treatment with low doses of DON, but not DON-3-Glu, DOM-1, or 3-epi-DON rapidly increased the phosphorylation of ERK1/2, p38, and/or JNK MAPKs in Caco-2 cells time- and dose- dependently, while the total MAPK level remained unchanged [24,37,[43], [44], [45]]. T-2 treatment dose-dependently phosphorylated p38 MARK in Caco-2 cells [46]. Reduced affinity of a trichothecene molecule for the ribosomal A-site due to modifications on the EPT scaffold may prevent the induction of RSR [37]. How trichothecenes initiate RSR in CECs is not thoroughly understood, but it is believed to be mediated by protein kinase R (PKR) or ZAKα (a MAP3K) as a ribotoxic stress sensor and initial signal transducer (Fig. 1A):

1)
PKR-mediated RSR

PKR is a ubiquitously expressed serine/threonine protein kinase which can be activated by double-stranded RNA (dsRNA). In human CECs treated with DON, activation of PKR was found to mediate the downstream events of MAPKs, such as upregulated production of the pro-inflammatory cytokine IL-8 [47] and downregulated production of mucus components [4,48], suggesting that DON activates MAPKs via PKR. It has been found also in other cell types that DON treatment rapidly activated PKR, which in turn activated MAPKs in the order JNK > p38 > ERK [49]. Zhou et al. [50] proposed a molecular model of DON-induced PKR activation. Multiple copies of PKR monomer constitutively associate at its dsRNA binding domains (DRBDs) with the ribosomal 18S and 28S rRNA, where PKR rapidly senses subtle perturbations in rRNA secondary and/or tertiary structure provoked by any binding of DON to ribosome. Such changes in rRNA structure bring adjacent PKR monomers in close proximity to allow dimerization, autophosphorylation, and activation of PKR [50]. As a ubiquitously expressed serine/threonine protein kinase, PKR can potentially phosphorylate a wide range of substrates in the cell. PKR is known to activate the MAP2Ks, e.g. MKK3/6 and MKK4 to activate p38 and JNK, respectively [51], and is also presumed to activate some upstream kinases of ERK1/2 to activate ERK1/2.

The activation of PKR by trichothecene is also known to phosphorylate eukaryotic initiation factor 2 (eIF2) to inhibit protein synthesis. eIF2 is a key mediator of initiation of protein synthesis, i.e. it binds to GTP, Met-tRNA, and the 40S ribosomal subunit to form the preinitiation complex (PC), which scans the mRNA from 5′ end to 3′ end for the first star codon (AUG). PC mediates the base pairing between the Met-tRNA and the start codon, and combines with the 60S ribosomal subunit to initiate translation, with the GTP hydrolyzed into GDP. The generated eIF2-GDP is then released and the GDP is replaced with GTP with the help of a guanine nucleotide exchange factor (GEF) to regenerate eIF2-GTP for a new round of protein synthesis. As DON-induced RSR, PKR gets activated and then phosphorylates eIF2 on its α subunit (eIF2α), which converts eIF2-GDP into a competitive inhibitor of its GEF. Consequently, formation of new PCs and overall frequency of mRNA translation are inhibited [52] (Fig. 1B).

2)
ZAKα-mediated RSR

ZAKα, as a MAP3K, has been found to perform the role as an independent ribotoxic stress sensor for DON and other ribotoxic agents. DON strongly induced ZAKα phosphorylation in human keratinocytes, resulting in a downstream pro-inflammatory response involving IL-1β secretion and NLRP1 inflammasome activation [53]. A molecular mechanism of ZAKα-mediated RSR has been proposed by Vind et al. [54] and further elucidated by Johansen et al. [55]. A ZAKα molecule consists of a N-terminal kinase domain (KD), a leucine zipper (LZ), a sterile α-motif (SAM) domain, and a ribosome binding region (RBR) containing a sensor domain and a C-terminal domain. ZAKα presumably binds to helix 14 of 18S rRNA exposed in the intersubunit space via the RBR, which senses any ribotoxic stress. The kinase activity of ZAKα is inhibited by the SAM domain until the RBR senses and recognizes ribotoxic stress signals [54,55]. When a trichothecene molecule binds to the ribosome, the resultant ribotoxic stress is presumed to relieve the inhibition of ZAKα by the SAM domain to allow autoactivation of ZAKα. This reduces affinity of the RBR with the ribosome and releases ZAKα as a cytoplasmic kinase to sequentially activate MAP2Ks and MAPKs (p38 and JNK) [54,55]. The role of ZAKα as an independent ribotoxic stress sensor has been demonstrated in different cell types for a variety of ribotoxic agents [53,55], but its role in trichothecene-induced RSR is yet to be experimentally verified in CECs.

PKR is an earlier known ribotoxic stress sensor, but the role of ZAKα as a ribotoxic stress sensor is attracting more recent interest. The hematopoietic cell kinase (HCK), a third well characterized ribotoxic stress sensor found in hematopoietic lineages [56], is not involved in RSR in CECs. How an CEC makes a choice between PKR and ZAKα upon trichothecene stimulation is still a question to answer. As consequence of trichothecene-induced RSR, the CECs undergo a variety of biological processes mediated by MAPK signaling, such as inflammation and apoptosis. MAPKs signaling leads to activation of pro-inflammatory transcription factors, preferentially by p38 and ERK 1/2 (See the section on inflammatory response) and/or to the phosphorylation of pro-apoptotic transcription factor p53, which subsequently activates the pro-apoptotic protein BAX. The induction of apoptosis appears to be more specifically driven by p38 and JNK than ERK1/2 [31,57].

Induction of endoplasmic reticulum stress (ERS) and unfolded protein response (UPR)

Endoplasmic reticulum (ER) has three transmembrane sensor proteins of ERS: PKR-like endoplasmic reticulum kinase (PERK), inositol requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6). In unstressed condition, these ERS sensors are kept inactive by their association with the ER chaperone BiP at their luminal domains [58]. Upon cellular exposure to DON or other ribotoxic stressors, protein synthesis is inhibited, resulting in aberrant accumulation of truncated peptides in ER [31]. BiP is preferentially associated to the unfolded or misfolded proteins, thus releasing the ERS sensors. Subsequently, PERK or IRE1α gets homodimerized and autophosphorylated for activation, and ATF6 is translocated to the Golgi apparatus for activation, thereby initiating UPR [58]. Among the three ERS sensors, PERK and IRE1α have been found to be activated by trichothecenes in CECs, and initiate UPR through the PERK/eIF2/ATF4 and Ire1α/XBP1 signaling pathways [[59], [60], [61]](Fig. 1C):

1)
UPR mediated by PERK/eIF2/ATF4 pathway

DON exposure significantly upregulated the phosphorylation of PERK and eukaryotic translation initiation factor 2 (eIF2), and the expression of BiP, activating transcription factor 4 (ATF4), and CCAAT/enhancer-binding protein homologous protein (CHOP) in mouse intestine, indicating the activation of PERK/eIF2/ATF4 pathway [61]. eIF2 is a key mediator of initiation of protein synthesis. Briefly, it binds to GTP, Met-tRNA, and the 40S ribosomal subunit to form the preinitiation complex (PC), which mediates the base pairing between the Met-tRNA and the start codon [52,62]. Under ERS caused by DON, PERK gets activated and then phosphorylates eIF2 on its α subunit (eIF2α) [61], which inhibits formation of new PCs and overall frequency of mRNA translation, thus relieving the ERS [52,62] (Fig. 1B). However, selective mRNAs that harbor upstream open reading frames, such as ATF4 mRNA, are preferentially translated in the presence of phosphorylated eIF2α. ATF4 can activate the transcription of multiple target genes, such as pro-inflammatory cytokines, and CHOP if ERS persists. CHOP is known as a pro-apoptotic transcription factor which can promote apoptosis by upregulating the levels of NOXA, BIM, and PUMA, and downregulating BCL-2 [58,63].

2)
UPR mediated by Ire1α/XBP1 pathway

Under ERS caused by trichothecenes, IRE1α gets activated to regain its endonuclease activity, with which IRE1α splices an intron from XBP1 mRNA (XBP1s), allowing its translation into a transcription factor that controls the expression of ER resident chaperones (BiP for example) and other enzymes responsible for restoring ER homeostasis, as well as some pro-inflammatory molecules [58,63]. Activation of IRE1α, XBP1s, and BiP by T-2 or DON has been observed in Caco-2 and HT-29 cells, as well as in mice intestinal tissue, confirming the activation of Ire1α/XBP1 pathway [59,60]. The activated IRE1α is also known to activate MAPKs and NF-κB. Briefly, the activated IRE1α recruits and activate TNF receptor-associated factor 2 (TRAF2), which then recruits apoptosis signal-regulating kinase (ASK1) and induces its activation by oligomerization and autophosphorylation. As an MAP3K, the activated ASK1 then launches the MAPK signaling cascade, resulting in activation of JNK and p38 MAPKs [59,62], which is known to mediate pro-inflammatory or pro-apoptotic response [31,57]. Meanwhile, the IRE1α-TRAF2 complex also recruits IKK to phosphorylate and degrade IκB, which coordinates with translational suppression of IκB by eIF2α, leading to NF-κB activation [64], but this is yet to be experimentally verified in trichothecene-exposed CECs.

To summarize, out of the three UPR signaling pathways (PERK, IRE1α, and ATF6 pathways), trichothecenes have been reported to activate PERK and IRE1α, but not ATF6 pathways in CECs, though potential role of ATF6 remains open. PERK can drive pro-inflammatory and pro-apoptotic responses via ATF4. Similarly, IRE1α can also mediate pro-inflammatory response (via MAPK or XBP1) or pro-apoptotic response (via MAPK). Both ATF4 and XBP1 also contribute to ERS relief. These signals can act additionally, synergistically, or antagonistically, cross-regulating the cell fate under trichothecene-induced ERS.

Induction of oxidative stress

Observations of trichothecene-induced oxidative stress

Oxidative stress is an important mode of toxicity of trichothecenes on CECs. Oxidative stress is caused by overload of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) free radicals, overwhelming the capacity of antioxidant system including glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), catalase, glutathione S-transferase (GST), and Heme Oxygenase 1 (HO-1), etc. [65,66]. Excessive ROS and RNS are known for their harmful interaction with macromolecules such as DNA, RNA, protein, and lipid, and organelles such as mitochondrion, and are believed to induce inflammation and apoptosis, reduce enterocyte proliferation, and interfere with epithelial renewal [67,68].

In HT-29 and Caco-2 cells, acute or chronic treatment with DON significantly increased production of ROS and nitrite (the stable oxidation product of nitric oxide (NO)) [19,45,[69], [70], [71]], quicky consuming the endogenous antioxidant GSH. As antioxidation response, activities of antioxidant enzymes including catalase, SOD, GPx, GST, and HO-1 were reported to be increased in different studies [45,69,70]. The antioxidation response was mediated by nuclear factor (erythroid-derived 2)-like 2 (Nrf2) [45,72], a transcription factor normally inhibited by kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm. Upon Keap1 oxidation by ROS, Nrf2 accumulates and migrates into the nucleus, where Nrf2 binds to antioxidant response elements (AREs) to activate the transcription of genes encoding antioxidant enzymes [65,72]. Generation of malondialdehyde (MDA), a biomarker of lipid peroxidation, was significantly increased by DON-induced oxidative stress in Caco-2 cells [23] and HT-29 cells [45]. Mitochondrial superoxide (O₂^•−) levels significantly increased in a human colon cancer cell line (HCT116) exposed to DON [73], indicating the origin of ROS. DON, DON-3-Sulf and DON-15-Sulf were all found to enhance the intracellular ROS level in HT-29 within the first 3 min of treatment, while both DON-sulfates lacked the ability to induce RSR [72], suggesting that ROS generation can be induced as a rapid cellular response to toxic stimulation independently of RSR. Duration of ROS generation varied from minutes [69,70,72] to hours [19,45,73], probably depending on dose and duration of toxin exposure, and rates of toxin metabolism and ROS clearance.

Mechanism of trichothecene-induced ROS production

Intracellular ROS can be generated by the mitochondrial electron transport chain (ETC), peroxisomes, NADPH oxidase, lipoxygenase, cyclooxygenases, and cytochrome P450s [74]. To date it is not well elucidated how trichothecenes induce overproduction of intracellular ROS, particularly in CECs. ROS generation is widely considered as consequence of mitochondrial stress [75]. In HT-29 cells exposed to DON or DON-sulfates, ROS was generated as an early response independent of RSR [72]. Considering most ROS in cells is produced by the mitochondrial ETC [76,77], the overproduction of ROS in CECs observed above is probably attributed to disturbance of mitochondrial ETC by trichothecenes. The DON-promoted mitochondrial superoxide generation in HCT116 cells suggested the critical role of mitochondria in trichothecene-induced ROS production [73].

Mitochondria are the powerplants of cells, producing energy in the form of ATP. The classical process of ATP production begins with glycolysis (in the cytosol), followed by pyruvate decarboxylation and tricarboxylic acid cycle (TCA cycle) (both in the mitochondrial matrix), generating NADH and FADH₂. The ETC, which is embedded in the mitochondrial inner membrane (IM), is composed of 4 enzymatic complexes, i.e. Complex Ⅰ (NADH dehydrogenase), Complex Ⅱ (succinate dehydrogenase), Complex Ⅲ (cytochrome bc1), and Complex Ⅳ (cytochrome c oxidase), IM-dissolved ubiquinone, and IM-flanking cytochrome c (Cytc). The electrons carried by NADH and FADH₂ enter the ETC via Complex Ⅰ and Complex Ⅱ, respectively, and are transported down the redox potential in the direction of ubiquinone/Complex Ⅲ/Cytc/Complex Ⅳ, and finally reduce oxygen molecules into water at the destination. The energy released during electron transport (i.e. oxidation) is used by Complexes Ⅰ, Ⅲ, and Ⅳ to pump protons from the mitochondrial matrix into the intermembrane space to establish and maintain a transmembrane electrochemical proton gradient (Δp), which forces protons back into the mitochondrial matrix through the transmembrane ATP synthase, driving the phosphorylation of ADP into ATP (a process known as oxidative phosphorylation). Under physiological conditions, 0.2–2 % of the electrons in the ETC are not transported normally to form water at the destination, but instead leak out of the ETC and react with oxygen to form superoxide, which can be further converted into other forms of ROS such as hydrogen peroxide [78]. This basal level of ROS is believed to have some physiological functions such as cellular signal transduction, but disturbance of the mitochondria, especially on their ETC by xenobiotics may increase electron leak and ROS generation to toxic levels.

The mitochondrion is probably another primary target for trichothecenes besides the ribosome. ROS overproduction, as an indicative of mitochondrial ETC dysfunction, is believed to be a primary toxic effect of trichothecenes, but can be exacerbated by other trichothecene-induced toxic mechanisms, such as pro-apoptotic signaling. Earlier study has indicated that trichothecenes can be rapidly absorbed by the mitochondria as early as 2 min, and change or even damage the normal structural morphology of animal mitochondria [68,79]. The mechanisms by which trichothecenes disturb the mitochondria ETC to generate ROS remains unclear, but presumably by impairing the activity of ETC components to increase electron leak (Fig. 1D). It was found in human and animal cells that T-2 could target and inhibit the activity of all the four mitochondrial ETC complexes and ATP synthase [[80], [81], [82]], inhibit oxygen consumption in the ETC [82,83], partially block electron flow between Complex Ⅲ or Cytc and O₂ [82], suppress ATP production, and increase intracellular ROS generation [81]. ATP production in Caco-2 cells was also suppressed dose-dependently by other trichothecenes such as DON, NX-2, and NX-3, but not by DOM-1 [1,43,71,84,85]. NADH dehydrogenase and succinate dehydrogenase form Complex Ⅰ and Complex Ⅱ of the ETC, respectively. Cytotoxicity assays based on mitochondrial dehydrogenases (e.g. CCK8 assay, MTS Assay, MTT assay, WST-1 assay, and Resazurin assay) showed that a variety of trichothecenes impaired mitochondrial dehydrogenase activity of CECs (Table 2). Although initiation of ROS production is considered as a rapid RSR-independent response to toxic stimulation, ROS production may possibly be prolonged or boosted by the early apoptotic events downstream of RSR. As pro-apoptotic agents, trichothecenes can activate the mitochondrial apoptosis pathway in CECs by promoting cytosolic release of mitochondrial Cytc (See the subsection on regulation of CEC viability). This probably depletes Cytc from the ETC, impairs the efficiency of electron transport, and elevates electron leak, resulting a dramatic increase of superoxide and hydrogen peroxide generation [86]. Taken together, the abovementioned changes in mitochondrial morphology, oxygen consumption, ATP production, mitochondrial dehydrogenase activity, and cytosolic release of mitochondrial Cytc indicate the potential ability of trichothecenes to disturb the mitochondrial ETC function and promote electron leak, which may account for the overproduction of ROS, but further study is needed to provide direct evidences and elucidate the underlying molecular mechanisms.

Table 2.

IC50 of trichothecenes on viability of human CECs.

Type	Trichothecene	Cell line	Cell status	Exposure duration	IC50	Assay	Reference
A	T-2	HCT116 WT	Not mentioned	48 h	24 nM	Methylene Blue Staining	[139]
	T-2	HCT116 FLIP	Not mentioned	48 h	7.5 nM	Methylene Blue Staining	[139]
	T-2	SW742	Proliferating	not mentioned	11.8 nM	Neutral red	[185]
	T-2	HT-29	Not mentioned	24 h	741 nM	CCK8	[186]
	T-2	Caco-2	Proliferating	24 h	14.83 µM	MTT	[152]
	T-2	Caco-2	Proliferating	24 h	12.92 µM	LDH	[152]
	T-2	Caco-2	Proliferating	24 h	842 nM	MTT	[59]
	T-2	Caco-2	Proliferating	24 h	15–30 nM	MTT	[138]
	T-2	Caco-2	Proliferating	72 h	13.37 nM	Resazurin	[135]
	T-2	Caco-2	Proliferating	72 h	23.1 nM	WST-1	[134]
	HT-2	Caco-2	Proliferating	72 h	45.9 nM	WST-1	[134]
	HT-2	Caco-2	Proliferating	72 h	47.44 nM	Resazurin	[135]
	DAS	Caco-2	Proliferating	72 h	17.74 nM	Resazurin	[135]
	Trichodermin	COLO201	Proliferating	24 h	123 μM	WST-1	[133]
	12-deoxytrichodermin	COLO201	Proliferating	24 h	420 μM	WST-1	[133]
	NX-3	HT-29	Proliferating	24 h	10–100 µM	Resazurin	[28]
	NX-3	HT-29	Proliferating	24 h	10–100 µM	SRB	[28]
	NX-3	HCEC-1CT	Proliferating	24 h	1–10 µM	Resazurin	[28]
	NX-3	HCEC-1CT	Proliferating	24 h	1–10 µM	SRB	[28]

B	DON	HT-29	Proliferating	24 h	10–100 µM	Resazurin	[28]
	DON	HT-29	Proliferating	24 h	10–100 µM	SRB	[28]
	DON	HCEC-1CT	Proliferating	24 h	1–10 µM	Resazurin	[28]
	DON	HCEC-1CT	Proliferating	24 h	1–10 µM	SRB	[28]
	DON	HCT116	Proliferating	24 h	70.49 µM	MTT	[141]
	DON	HCT116	Not mentioned	24 h	125 µM	FDA	[73]
	DON	HCT116	Proliferating	24 h	150 µM	FDA	[146]
	DON	HT-29	Not mentioned	24 h	72.39 µM	MTT	[60]
	DON	HT-29	Proliferating	24 h	10 μM	MTT	[99]
	DON	HT29-16E	Differentiated	48 h	79 µM	LDH	[4]
	DON	HT29-16E	Differentiated	48 h	88 µM	MTT	[4]
	DON	HT29-16E	Differentiated	48 h	≈100 µM	LDH	[48]
	DON	HT29-16E	Differentiated	48 h	10–100 µM	MTT	[48]
	DON	HT-29	Proliferating	24 h	10–25 µM	SRB	[27]
	DON	HCEC-1CT	Proliferating	24 h	1–10 µM	SRB	[27]
	DON	Caco-2	Proliferating	24 h	≈10 µM	SRB	[27]
	DON	Caco-2	Proliferating	24 h	16.32 µM	MTT	[126]
	DON	Caco-2	Proliferating	48 h	2.88 µM	CellTiter-glo	[84]
	DON	Caco-2	Proliferating	72 h	2.9 µM	MTT	[187]
	DON	Caco-2	Proliferating	48 h	≈ 4 µM	WST-1	[188]
	DON	Caco-2	Proliferating	24 h, 72 h	10 μM (24 h), 4.3 μM (72 h)	MTS	[97]
	DON	Caco-2	Differentiated	24 h, 72 h	>10 μM (24 h, 72 h)	MTS	[97]
	DON	Caco-2	Proliferating	24 h, 72 h	3.7 μM (24 h, 72 h)	Neutral red	[97]
	DON	Caco-2	Differentiated	24 h, 72 h	>10 μM (24 h, 72 h)	Neutral red	[97]
	DON	Caco-2	Not mentioned	24 h, 48 h, 72 h	21.94 µM (24 h), 9.39 µM (48 h), 6.18 µM (72 h)	CCK8	[144]
	DON	Caco-2	Proliferating	24 h	8.5 μM	CCK8	[156]
	DON	Caco-2	Proliferating	24 h	1.81 µM	CCK8	[19]
	DON	Caco-2	Proliferating	48 h	2.23 µM	MTT	[44]
	DON	Caco-2	Not mentioned	72 h	21.5 µM	Neutral red	[23]
	DON	Caco-2	Not mentioned	72 h	25 µM	MTT	[23]
	DON	Caco-2	Proliferating	24 h	10–100 µM	Trypan blue exclusion	[22]
	DON	Caco-2	Proliferating	72 h	1 µM	WST-1	[134]
	DON	Caco-2	Proliferating	48 h	1.30 µM	CellTiter-glo	[37]
	DON	Caco-2	Proliferating	48 h	1–3 µM	CellTiter-glo	[43]
	DON	Caco-2	Differentiated	8d	30–100 µM	CellTiter-glo	[43]
	DON	Caco-2	Proliferating	24 h, 48 h, 72 h	5.07 μM(24 h), 2.62 μM(48 h), 1.95 μM(72 h)	CCK8	[71]
	DON	SW480	Proliferating	24 h, 48 h	≈6.75 μM (24 h, 48 h)	CCK8	[149]
	DON	Caco-2	Proliferating	48 h	1.39 µM	MTT	[136]
	DON	Caco-2	Proliferating	48 h	1.19 µM	Neutral red	[136]
	DON	Caco-2	Proliferating	24 h, 48 h, 72 h	6.17 µM (24 h), 3.86 µM (48 h), 1.46 µM (72 h)	CCK8	[100]
	DON	COLO201	Proliferating	24 h	86 μM	WST-1	[133]
	DOM-1	COLO201	Proliferating	24 h	>700 μM	WST-1	[133]
	3ADON	Caco-2	Proliferating	48 h	2.94 µM	MTT	[136]
	3ADON	Caco-2	Proliferating	48 h	1.99 µM	Neutral red	[136]
	3ADON	Caco-2	Proliferating	24 h, 48 h, 72 h	13.19 µM (24 h), 10.81 µM (48 h), 7.84 µM (72 h)	CCK8	[100]
	15ADON	Caco-2	Proliferating	24 h, 48 h, 72 h	3.86 µM (24 h), 2.33 µM (48 h), 1.44 µM (72 h)	CCK8	[100]
	15ADON	Caco-2	Proliferating	48 h	1.47 µM	MTT	[136]
	15ADON	Caco-2	Proliferating	48 h	1.1 µM	Neutral red	[136]
	FusX	Caco-2	Proliferating	48 h	0.02 µM	Neutral red	[136]
	FusX	Caco-2	Proliferating	48 h	0.04 µM	MTT	[136]
	FusX	Caco-2	Differentiated	3 h, 24 h, 72 h	>5μM (3 h, 24 h), 1.96 μM (72 h)	Neutral red	[98]
	FusX	Caco-2	Proliferating	3 h, 24 h, 72 h	>5μM (3 h), 0.4 μM (24 h), 0.1 μM (72 h)	Neutral red	[98]
	FusX	Caco-2	Differentiated	3 h, 24 h, 72 h	>5μM (3 h, 24 h, 72 h)	MTS	[98]
	FusX	Caco-2	Proliferating	3 h, 24 h, 72 h	>5μM (3 h), 0.68 μM (24 h), 0.35 μM (72 h)	MTS	[98]
	NIV	Caco-2	Proliferating	3 h, 24 h, 72 h	>10 μM (3 h, 24 h, 72 h)	MTS	[98]
	NIV	Caco-2	Differentiated	3 h, 24 h, 72 h	>10 μM (3 h, 24 h, 72 h)	MTS	[98]
	NIV	Caco-2	Proliferating	3 h, 24 h, 72 h	>10 μM (3 h), 2.94 μM (24 h), 1.39 μM (72 h)	Neutral red	[98]
	NIV	Caco-2	Differentiated	3 h, 24 h, 72 h	>10 μM (3 h, 24 h, 72 h)	Neutral red	[98]
	NIV	Caco-2	Proliferating	48 h	0.9 µM	MTT	[136]
	NIV	Caco-2	Proliferating	48 h	0.69 µM	Neutral red	[136]
	NIV	Caco-2	Proliferating	72 h	0.5 µM	WST-1	[134]
	TCN	HT-29	Proliferating	72 h	≈1.2 µM	SRB	[29]
	TCN	HCT116	Proliferating	72 h	0.25 µM	SRB	[29]

D	VJ	SW620	Proliferating	24 h	180 nM	MTT	[142]
	VJ	HCT116	Proliferating	24 h	200 nM	MTT	[142]
	VJ	HCT116	Proliferating	48 h	12.71 nM	CCK8	[137]
	VL	HCT116	Proliferating	48 h	9.72 nM	CCK8	[137]
	Mytoxin F	HCT116	Proliferating	48 h	5.8 nM	CCK8	[137]
	Mytoxin G	HCT116	Proliferating	48 h	1782.33 nM	CCK8	[137]
	RJ	HCT116	Proliferating	48 h	8.5 nM	CCK8	[137]
	RJ	COLO201	Proliferating	24 h	42 μM	WST-1	[133]
	12-deoxyroridin J	COLO201	Proliferating	24 h	50 μM	WST-1	[133]
	Mytoxin D	HCT116	Proliferating	48 h	3.12 nM	CCK8	[137]
	Mytoxin E	HCT116	Proliferating	48 h	44.43 nM	CCK8	[137]
	RH	COLO201	Proliferating	24 h	82 μM	WST-1	[133]
	8α-hydroxyroridin H	COLO201	Proliferating	24 h	51 μM	WST-1	[133]
	8α-acetoxyroridin H	COLO201	Proliferating	24 h	46 μM	WST-1	[133]
	8α-acetoxyroridin H	HCT116	Proliferating	48 h	87.28 nM	CCK8	[137]
	Epiisororidin E	COLO201	Proliferating	24 h	47 μM	WST-1	[133]
	12-deoxyepiisororidin E	COLO201	Proliferating	24 h	51 μM	WST-1	[133]
	Satratoxin H	Caco-2	Proliferating	72 h	10.5 nM	WST-1	[134]
	Satratoxin G	Caco-2	Proliferating	72 h	12.1 nM	WST-1	[134]

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Activation of NF-κB by ROS

ROS is well known for its ability to regulate activation of NF-κB, an important transcription factor involved in a variety of biological functions such as inflammation, immune response, cell survival, and metabolism [87,88]. Overproduction of intracellular ROS induced by DON in HT-29 cells was found to be associated with activation of NF-κB. The nuclear translocation of active NF-κB occurs as early as 15 min following DON exposure [45,69,70]. NF-κB is a heterodimer typically composed of p50 and p65 subunits. In the latent condition, NF-κB stays inactive in the cytosol by being bound to its inhibitor IκB. ROS regulates NF-κB pathway by complex mechanisms, depending on cell type, stimulation phase (i.e. early/late), and subcellular location (i.e. cytoplasmic/nuclear), etc. [64,89]. As an early response to mitochondrial ROS induced by trichothecenes in CECs, activation of NF-κB is probably mediated by the Src non-receptor tyrosine kinase as the most relevant mechanism. Indeed, activation of Src by oxidative stress has been demonstrated in Caco-2 cells [90]. Src maintains an autoinhibited conformation in the latent condition. When cytoplasmic ROS accumulates, H₂O₂ activates Src by oxidizing Cys185 and Cys277 of Src to induce conformational changes which destruct the autoinhibition, allow for autophosphorylation at Tyr416, and expose binding sites for downstream substrates. In addition, inactivation of protein tyrosine phosphatases (PTP), the inhibitors of Src, by oxidation of their catalytic cysteines also facilitates the activity of Src [91]. The activated Src then activates NF-κB signaling by phosphorylating IκB indirectly (via Src/PKD1/IKK/IκB/NF-κB axis) or directly (via Src/IκB/NF-κB axis) (Fig. 1E):

1)
NF-κB activation by Src/PKD1/IKK/IκB/NF-κB axis. The activated Src activates another non-receptor tyrosine kinase Abl and protein kinase Cδ (PKCδ). Subsequently protein kinase D1 (PKD1) is phosphorylated at Tyr95 (binding site of PKCδ) by Src, at Tyr463 (pleckstrin homology (PH) domain) by Abl, and at Ser738 and Ser742 (kinase domain) by PKCδ to be fully activated [92]. The activated PKD1 then phosphorylates the IKKβ subunit of IKK complex (NEMO/IKKα/IKKβ), which then mediates Ser32/Ser36 phosphorylation, ubiquitination and proteasomal degradation of IκB, resulting in the release and nuclear translocation of active NF-κB [64].
2)
NF-κB activation by Src/IκB/NF-κB axis. Src can also directly phosphorylate IκB at Tyr42 independently of IKK, leading to IκB dissociation (but not degradation), followed by NF-κB activation and nuclear translocation [64].

The NF-κB pathway activated by ROS is believed to mediate the inflammatory response in CECs stimulated by DON and other trichothecenes (See the subsection on inflammatory response).

Mechanism of trichothecene-induced RNS production

NO is produced by oxidation of L-arginine into L-citrulline, catalyzed by nitric oxide synthases (NOSs), which include neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). nNOS and eNOS are constitutively expressed in the nervous system and endothelium, respectively, while iNOS is found in IECs and many other cell types and is expressed only by induction [93,94]. This makes iNOS more relevant to trichothecene-induced RNS production in CECs than nNOS and eNOS. iNOS expression is mainly regulated by NF-κB [93] (Fig. 1F). Indeed, intracellular RNS induced by DON in HT-29 cells was found to be associated with activation of NF-κB [69,70]. In response to trichothecene exposure, NF-κB can be activated by ROS (see the previous paragraphs), UPR (See the section on ERS and UPR), and/or pro-inflammatory cytokines (see the subsection on inflammatory response) in CECs. The activated NF-κB then translocates into the nucleus, where its transcriptional activity is increased by MSK [95], and interacts with the iNOS gene promoter to trigger iNOS transcription. Besides the NF-κB pathway, iNOS expression can also be induced by interferon‐γ (INF-γ) and mediated by JAK/STAT1α pathway in other cells, but this has not yet been evidenced in trichothecene-treated CECs. Once expressed, iNOS catalyzes the formation of NO until iNOS is degraded. Interestingly, it has been also reported that DON treatment time- and dose- dependently increased iNOS mRNA but decreased iNOS protein and NO production in Caco-2 cells [96]. This suggests that the net influence of DON on NO generation is probably subject to both the promotion of iNOS transcription (mediated by NF-κB) and the inhibition of iNOS protein synthesis (as a result of ribotoxic stress and UPR). The effect of NO on cells is concentration-dependent. Specifically, an appropriate concentration facilitates immune response, while an inappropriately high concentration exhibits toxic effects. NO can also be converted into peroxynitrite (ONOO⁻), another RNS free radical, by reacting with superoxide [93].

Induction of DNA damage and DNA damage response

Induction of DNA damage

Trichothecenes have been found both in vitro and in vivo to induce DNA damage in CECs, even at low concentrations compatible with actual exposure, indicating potential genotoxicity. DON, NIV, and FusX at realistic sub-cytotoxic doses have been demonstrated by comet assay to induce DNA damage time- and dose-dependently in dividing and/or differentiated Caco-2 cells [97,98]. DNA fragmentation was also observed in HT-29 cells exposed to DON at a higher dose [99]. Transcriptomic analysis on Caco-2 cells treated with DON, 3-ADON or 15-ADON revealed that ataxia-telangiectasia mutated kinase (ATM) was transcriptionally activated, indicating the DNA damaging property of these trichothecenes [100]. In in vivo trial with mice, DNA damage appeared in the colon 8 h after oral or intraperitoneal dosing of NIV, possibly as a result of systemic absorption [101].

Both ROS-independent and ROS-dependent manners of DNA damage caused by DON have been proposed in different studies. It was reported that DON exposure caused DNA damage in human gastrointestinal epithelial cells without ROS generation [99,102]. Indeed, DON is able to enter the nucleus by simple diffusion, and probably cause DNA damage by direct interaction [102,103]. By contrast, other studies suggested that exposure to trichothecenes leads to the production of ROS, which in turn binds to DNA base fragments and cause changes in DNA structure, eventually resulting in DNA damage [66,104]. Among the well-documented DNA damage types caused by oxidative stress, the production of 8-Hydroxydeoxyguanosine (8-OHdG), an oxidized derivative of guanine, is of particular interest. Guanine is particularly vulnerable to ROS-induced modifications due to its low oxidation potential [65]. It was also proposed that free cytoplasmic nucleotides are oxidized by mitochondrial ROS before incorporated into DNA and RNA [105]. Since both ROS-dependent and ROS-independent DNA damage have been observed in different cells, it may suggest that DNA damage is likely caused by multiple mechanisms: either indirectly through DON-induced ROS or directly by DON itself. Exposure conditions (e.g. dose and duration) and cellular characteristics (e.g. nuclear membrane permeability, mycotoxin metabolism efficiency, and ROS-scavenging capacity) may differentially influence the effects of trichothecenes and ROS, and thus determine the primary pathway of DNA damage. Consequently, the induction of DNA damage by DON or ROS may occur independently or concurrently. Further investigation is required to clearly understand the molecular mechanisms underlying trichothecene-induced DNA damage.

Induction of DNA damage response

Cells with severe DNA damage tend to be removed through apoptosis to prevent mutation from being passed down. As DNA damage response (DDR), the cells undergo cell cycle arrest or apoptosis, under the regulation of ATM and p53 as key signal transducers (Fig. 1G). ATM is a serine/threonine protein kinase and works as an initial signal transducer of DDR. ATM exists as inactive dimers in cells until activated by DNA damage, typically by double-strand break (DSB). Once DNA damage occurs, it is detected by the sensor MRE11-RAD50-NBS1 (MRN) complex, which is recruited to the DBS sites and subsequently recruits ATM via its ATM-binding motif. Upon recruitment by MRN complex, ATM is activated by acetylation and autophosphorylation for full kinase activity, leading to ATM monomerization [106]. The activated ATM monomer then activates the transcription factor p53. In an unstressed cell, p53 is maintained in low level in the nucleus as an inactive format by its negative regulator MDM2, which binds to p53 to block its action and transports it to the cytosol for proteasomal degradation. ATM activated by DNA damage activates p53 by phosphorylating MDM2 and p53, which disassociates MDM2 from p53 to avoid p53 degradation, leading to a quick accumulation of p53 in the nucleus. The activation of p53 initiates transcription of target genes including the cell cycle inhibitor p21 or pro-apoptotic factors such as Bax and Fas/Fas ligand [106,107]. DON has been found to induce DNA damage in human CECs, with upregulation of ATM mRNA [100] and activation of p53 and caspase-3 [99]. Activation of ATM/p53 signaling pathway by DON-induced DNA damage was also substantiated in murine gastrointestinal epithelial cells [102].

Modulation of inflammatory response

Modulation of inflammatory markers

Trichothecenes are widely known for their pro-inflammatory property, so are considered risk factors contributing to intestinal inflammatory processes. Trichothecene-induced inflammation in CECs is marked by the production of pro-inflammatory cytokines and other pro-inflammatory molecules. For example, IL-8 (also known as CXCL8) is considered an early marker of inflammatory process and has been demonstrated to be a particularly potent chemo-attractant for leukocytes and T-lymphocytes underlying IECs [108]. In vitro studies showed that trichothecenes such as T-2 [46], NX-3, DON [24,45,72,[108], [109], [110], [111], [112], [113], [114]], 3ADON and 15ADON [[115], [116], [117], [118]] induced transcription, synthesis and secretion of pro-inflammatory cytokines IL-8, IL-6, TNF-α and/or IL-1β in the colonic epithelial cell lines Caco-2, HT-29, HCEC-1CT, and others. In addition to these pro-inflammatory cytokines, DON also upregulated the expression of cyclooxygenase-2 (COX-2) to catalyze the synthesis of prostaglandin E2 (PGE-2), which mediates inflammatory processes by regulating vascular dilatation, mucosal secretion, and fever generation [45,69,70,72,112]. Both chronic and acute DON exposure can induce inflammation in both differentiated and proliferating Caco-2 cells. Upregulation of IL-8 secretion and PGE-2 synthesis upon acute exposure to DON were found to be dose-dependent [112]. The pro-inflammatory property of DON was confirmed in in vivo trial with rats, where ingestion of DON increased the pro-inflammatory markers TNF-α and IL-17 in the colon [15]. Surprisingly, DON also reportedly downregulated IL-1β, IL-8, and CCL2 transcription in Caco-2 cells [119], suggesting anti-inflammatory potential of DON governed by unknown mechanisms.

Trichothecenes not only initiates inflammation but also exacerbates inflammatory signs in CECs, as observed in in vitro and in vivo studies, and also at both mRNA and protein levels. Pre-treatment with DON upregulated mRNA levels of IL-8, TNF-α and IL-1β in Caco-2 cells inflamed with IL-1β [120]. T-2 [46] and DON [47,111] upregulated the secretion of IL-8 in inflamed Caco-2 cells pre-stimulated with IL-1β, TNF-α or LPS. DON at subtoxic levels exacerbated colitis in mice, as demonstrated by increased disease activity index, decreased colon length, increased morphological damage, increased IL-1β and TNF-α expression, and decreased the anti-inflammatory cytokine IL-10 expression [121]. Chronic exposure to DON also worsened the colitis-associated colorectal cancer (CRC) in mice, as shown by increases in colitis scores, tumor grades, and histological hyperplasia [122].

Besides modulating the expression and release of pro-inflammatory cytokines and COX-2, DON can also modify the local immune response in the large intestine by changing the production of receptors of cytokines [71], iNOS/NO [69,70,96], toll-like receptors (TLRs) [123], and host defense peptides (HDPs) [124] in human or animal models. This may lead to changes in the composition and functions of gut microbiota and susceptibility to infection. It is noteworthy that DON and other trichothecenes may also modulate the inflammatory response of the resident immune cells (e.g. macrophages, dendritic cells) in the lamina propria [125]. Together with CECs, these immune cells collectively orchestrate the inflammation response in the colonic mucosa.

The pro-inflammatory effects of trichothecenes on CECs are apparently structure-dependent. Presence or absence of a C8-carbonyl group on of the EPT scaffold seems to have little influence on the pro-inflammatory potential of trichothecenes. NX-3 (without a C8-carbonyl moiety) and DON (with a C8-carbonyl moiety) showed comparable potency with respect to immunomodulatory and pro-inflammatory potential [113]. Acetylation on C3 or C15 of EPT scaffold significantly modified the pro-inflammatory property of DON, with the ability to produce IL-8 on Caco-2 cells ranked as 3ADON < DON < 15ADON [118]. DON-3-Glu, DON-sulfates, and the de-epoxidized mycotoxin DOM-1 had no effect on IL-8 secretion or COX-2 [72,117]. Extensive investigation is necessary to fully understand how molecular structure, specifically the substitutional groups within the EPT scaffold influences the pro-inflammatory potential of trichothecenes.

Mechanisms of modulation of inflammatory response

The inflammatory responses induced by trichothecenes in CECs have been found to be mediated by MAPK, NF-κB, JAK/STAT, PERK/eIF2/ATF4, and IRE1α/XBP1 pathways, among which MAPK and NF-κB pathways are believed to be crucial [25,112,126] (Fig. 1F), and inflammation is sustained by positive feedbacks of these signal pathways.

1)
Inflammation mediated by MAPK pathways

Both in vitro and in vivo studies have exhibited that trichothecenes induces inflammation in CECs via the mediation of MAPKs (Fig. 1A,C,F,O). Application of MAPK inhibitors to Caco-2 or HT-29 cells exposed to T-2 or DON identified MAPKs as the mediators of the production of IL-8, and COX-2/PGE-2. Specifically, p38 and ERK1/2 play more significant roles in these trichothecene-induced inflammatory responses than JNK does [24,46,47,112,114]. Chronic exposure to DON worsened colitis-associated CRC of mice in company with upregulated expression of genes for MAPK signaling including the MEK1/2 MAP2K and the ERK1/2 MAPK [122]. MAPKs can be activated by trichothecenes via RSR (See the subsection on RSR), UPR (See the section on ERS and UPR), EGFR [114], and probably other toxic effects directly or indirectly resulting from trichothecene exposure. Among these MAPK activation mechanisms, RSR and EGFR have been directly evidenced to be responsible for the trichothecene-induced inflammatory response in CECs. Inflammation is considered the first major consequence of RSR [31]. Indeed, IL-8 production induced by DON depends largely (although not entirely) on the activation of PKR, a RSR mediating kinase, which activates its downstream MAPKs [47]. Another study revealed that DON-induced IL-8 transcription was mediated partially by EGFR-activated ERK, which upregulated the expression of Egr1, the key transcriptional factor directly responsible for IL-8 gene expression in CECs [114]. MAPKs are known to mediate the inflammatory response also by activating other inflammation-related transcription factors such as AP-1 complex, ATF2, C/EBPβ, CHOP, CREB, Ets-1, and Elk-1, which work dependently or independently of NF-κB, to promote expression of genes of inflammation mediators such as pro-inflammatory cytokines and COX-2 [[127], [128], [129], [130], [131]].

2)
Inflammation mediated by PERK/eIF2/ATF4 and IRE1α/XBP1 pathways

Trichothecenes can induce ERS and the subsequent UPR to activate the PERK/eIF2/ATF4 and IRE1α/XBP1 signaling pathways, as demonstrated in human CECs and in mice intestine (See the section on ERS and UPR). As transcription factors, ATF4 and XBP1 promote expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α through direct binding to their promoters, and decrease the production of anti-inflammatory cytokine IL-10 in epithelial cells [59,63] (Fig. 1C,F).

3)
Inflammation mediated by NF-κB pathway

NF-κB is a potent pro-inflammatory transcription factor. Its activation can increase the expression of inflammatory mediators, such as pro-inflammatory cytokines and COX-2 [111]. It was evidenced that NF-κB mediated the NX-3 or DON-induced expression of TNF-α, IL-1β, IL-8, IL-6, and/or COX-2/PGE-2 in Caco-2, HT-29, and HCEC-1CT cells [47,69,70,108,[111], [112], [113]]. As response of CECs to trichothecene exposure, NF-κB can be activated by ROS (see the subsection on ROS-activated NF-κB), UPR (See the section on ERS and UPR), and/or proinflammatory cytokines (e.g. TNF-α and IL-1β) produced in MAPK or UPR signaling (See previous paragraphs) (Fig. 1C,E,P). TNF-α and IL-1β have been well elucidated as activators of NF-κB pathway. Briefly, TNF-α and IL-1β bind to their receptors (TNFR and IL-1R, respectively) on the cytoplasmic membrane, and recruit and activate the TAK1/TAB kinase complex through their respective TRAF complexes. The IKK complex is activated by TAK1/TAB through phosphorylation of IKKβ at Ser177 and Ser181, and then phosphorylates IκB, leading to release and nuclear translocation of NF-κB, which serves as transcription factor [87]. The transcriptional activity of NF-κB is regulated by mitogen- and stress-activated kinase (MSK), a downstream substrate activated by ERK1/2 and p38 MAPKs. MSK can directly increase transcriptional activity of NF-κB through phosphorylation of the p65 subunit at Ser276, which is required for most p65-dependent transcription. MSK also phosphorylates the histone H3 at Ser10 in NF-κB-dependent promoters to increase DNA accessibility for NF-κB binding [95]. The fully activated NF-κB activates the transcription of genes encoding COX-2 and pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, IL-12, IL-23, and TNF-α [87] (Fig. 1F). Specific inhibition against p38 or ERK was found to abolish IL-8 production in DON-treated Caco-2 cells [47,112], probably due to the loss of MSK-induced transcriptional activity of NF-κB.

4)
Inflammation mediated by JAK/STAT pathway

JAK/STAT is another pathway involved in DON-enhanced inflammation (Fig. 1F,H). The exacerbation of established colitis in mice by DON was mediated by JAK/STAT pathway, as indicated by enhanced JAK2 and STAT3 phosphorylation, while intraperitoneal injection of JAK2 inhibitor attenuated this exacerbation [121]. The JAK2/STAT3 pathway can be activated by some pro-inflammatory cytokines (e.g. IL-6) produced via MAPK, UPR and/or NF-κB signaling. When these ligands bind to their receptors on the cell membrane, the receptors get dimerized, followed by autophosphorylation and activation of JAK, which recruits and activates STAT by phosphorylation. STAT then gets dimerized and translocates from the cytoplasm into the nucleus to function as a transcription factor for inflammation-mediating genes [132].

Regulation of CEC viability

Influence of trichothecenes on CEC viability

The general cytotoxicity of trichothecenes on CECs is indicated by their effects on cell viability. IC50 values of different trichothecenes on viability of colonic epithelial cell lines are summarized in Table 2. The cytotoxicity of trichothecenes is subject to 1) the molecular structure of a trichothecene, 2) dose of exposure, and 3) the differentiation status of cells:

1)
The molecular structure of trichothecenes

Interactions between trichothecenes and the ribosome depend on the molecular structure of trichothecenes. Modifications on the EPT scaffold are supposed to affect these interactions and the potential cytotoxicity (See the subsection on inhibition of protein synthesis). The C4,C15-macrocyclic ring, C12,C13-epoxide ring, acetylation, hydroxylation, and glucose or sulfate conjugation have been documented to affect the cytotoxicity of trichothecenes on CECs:

Macrocyclic trichothecenes are generally more toxic than non-macrocyclic trichothecenes on CECs. Macrocyclic trichothecenes RJ, RH, epiisororidin E, 8α-hydroxyroridin H, and 8α-acetoxyroridin H were found to have lower IC50 values than their non-macrocyclic counterparts DON and trichodermin on the viability of COLO201 cells [133]. Similarly, IC50 values of macrocyclic trichothecenes satratoxins H and G on viability of Caco-2 cells were also lower than those of T-2, HT-2, DON, and NIV [134]. It is believed that the C4,C15-macrocyclic ring makes the most critical contribution to the cytotoxicity of macrocyclic trichothecenes.

The C12,C13-epoxide ring is critical for the cytotoxicity of non-macrocyclic trichothecenes on CECs, and de-epoxidation results in detoxification. DON and trichodermin, each with an intact C12,C13-epoxide ring, had much lower IC50 values on the viability of COLO201 cells than their respective de-epoxidized counterparts DOM-1 and 12-deoxytrichodermin [133]. In Caco-2 cells, DOM-1 lacked the ability to impair cell viability as compared with DON [37,117]. NX-3 was more toxic to HT-29 and HCEC-1CT cells than its de-epoxidized derivative NX3-M1 [28]. For macrocyclic trichothecenes like RJ and epiisororidin E, de-epoxidation caused only minor reduction in their cytotoxicity [133].

Acetylation at different sites of the EPT scaffold can markedly influence the cytotoxicity of trichothecenes. Trichothecenes with a C4-acetyl group, such as T-2, DAS, and FusX exhibited lower IC50 values on the viability of Caco-2 cells than their non C4-acetylated counterparts, i.e. HT-2 (compared with T-2 and DAS) [134,135], DON and NIV (compared with FusX) [98,136]. These data suggest that acetylation at C4 potentiates the cytotoxicity of trichothecenes. 15ADON had a similar or lower IC50 than DON on the viability of Caco-2 cells [100,136], suggesting that C15-acetylation tends to preserve or augment the cytotoxicity of DON. By contrast, acetylation at C3 diminishes the cytotoxicity of trichothecenes. DON exhibited substantially higher cytotoxicity than 3ADON, as evidenced by the higher IC50 value of 3ADON on the viability Caco-2 cells [100,136]. Similarly, NX-3 also exhibited higher cytotoxicity on Caco-2 cells than its C3-acetylated derivative NX-2, but this difference was less pronounced than that between DON and 3ADON [85]. For macrocyclic trichothecenes, the effect of acetylation at C8 on cytotoxicity appears irregular. RH was less toxic to COLO201 cells than its C8-acetylated derivative 8α-acetoxyroridin H, as evidenced by the higher IC50 of RH [133], while mytoxin E, the C8-acetylated form of RJ, had a 5-fold greater IC50 value than RJ on the viability of HCT116 cells, indicating weakened cytotoxicity [137].

Hydroxylation at different sites increases the cytotoxicity of both non-macrocyclic and macrocyclic trichothecenes on CECs, but to a smaller extent than acetylation does at the same sites. For examples, NIV and FusX are the C4-hydroxylated variant and C4-acetylated variant of DON, respectively. When tested on Caco-2 cells, NIV showed lower IC50 values on cell viability than DON but higher IC50 values than FusX [98,134,136]. HT-2, which has hydroxyl group at C4, exhibited higher IC50 values on the viability of Caco-2 cells than T-2, which has an acetyl group at C4 [134,135]. Hydroxylation enhances the cytotoxicity of macrocyclic trichothecenes also. VL (C8-hydroxylated derivative of VJ), mytoxin D (C3-hydroxylated derivative of RJ), mytoxin E (8α-acetoxyroridin H derivative with a hydroxylated macrocyclic ring), and mytoxin F (VJ derivative with a hydroxylated macrocyclic ring) had lower IC50 values than their respective parent compounds on the viability of HCT116 cells [137]. 8α-hydroxyroridin H (the C8-hydroxylated variant of RH) showed a lower IC50 value on the viability of COLO201 cells than RH, but a higher IC50 value than 8α-acetoxyroridin H (C8-acetylated variant of RH) [133]. Contrary to DON, 3-epi-DON did not impair the viability of Caco-2 cells [37], suggesting that the configuration of the C15-(R)-hydroxyl group is critical for the cytotoxicity of DON and probably other trichothecenes on CECs.

Glucose or sulfate conjugation significantly reduces the cytotoxicity of both non-macrocyclic and macrocyclic trichothecenes on CECs. DON-3-Glu, a glycosylated variant of DON, was less toxic to both proliferating and differentiated Caco-2 cells than DON [43,117]. DON-3-Sulf and DON-15-Sulf even sustained a distinctive proliferative stimulus on human HT-29 cells [27,72]. In silico analysis revealed that DON-3-Glu and DON-sulfates, contrary to DON, was unable to bind to the A-site of the ribosome peptidyl transferase center, the main target for DON toxicity [43,72]. Detoxification by glycosylation was also observed in macrocyclic trichothecenes. Mytoxin G, as the C3-glycosylated form of VJ, demonstrated a 140-fold higher IC50 value than VJ on the viability of HCT116 cells, indicating loss of cytotoxicity [137].

2)
The dose of exposure

Trichothecenes affect viability of CECs dose-dependently [22,37,84,120,[138], [139], [140], [141], [142]], and hormesis may occur at low doses. DON, NX-2, and NX-3 increased cell viability of HT-29 and Caco-2 cells to different extents at low doses but dramatically suppressed it at high doses [27,85,143]. The increased cell viability by low doses of DON was associated with enhancement of metabolism [143]. T-2 at low doses did not significantly affect the viability of HCT116 cells, but enhanced the colony formation and cell migration, while high doses suppressed cell viability [139]. Mixtures of multiple trichothecenes also showed dose-dependent cytotoxicity on CECs. Binary or ternary combination of DON with 3ADON and/or 15ADON exhibited synergistic cytotoxicity on Caco-2 cells at concentrations <IC50 and additive cytotoxicity at concentrations ≥IC50. Similarly, binary combinations of DON with NIV or FusX showed synergistic cytotoxicity at low doses and additive cytotoxicity at high doses; in contrast, ternary combination of DON-NIV-FusX showed antagonistic cytotoxicity [136].

3)
The differentiation status of cells

Proliferating cells are more sensitive to trichothecenes than differentiated cells. DON treatment impaired the viability of Caco-2 cells [1,97] and HT-29 cells [26] more pronouncedly in the proliferating status than in the differentiated status. Transcriptome analysis revealed that the number of differentially expressed genes and the amplitude of the effect of DON were higher in proliferating cells than differentiated cells. The differentiation process of proliferative cells was delayed, as indicated by the delayed establishment of transepithelial electrical resistance (TEER) [1]. Similarly, NIV and FusX also showed higher cytotoxicity on proliferating cells than differentiated cells, as indicated by the differences in IC50 value on the viability of Caco-2 cells [98].

Mechanisms of regulation of CEC viability

Cell survival or death is a result of death and survival signaling competition. The reduction in cell viability results from inhibited cell proliferation and/or enhanced cell death. Trichothecenes have been well documented on their ability to regulate cell proliferation and cell death. Apoptosis is the best studied regulated cell death (RCD) induced by trichothecenes in CECs, while potential roles of other forms of RCD such as autophagy-dependent cell death (ADCD), necroptosis, ferroptosis, and pyroptosis await further investigation. In vitro acute or chronic treatment with trichothecenes DON, TCN, VJ, mytoxin D, or mytoxin F, has been reported to cause apoptosis in human colonic epithelial cell lines Caco-2 [19,71,97,126,144], HT-29 [26,45,69,70,99,145], HCT116 [29,73,137,142,145,146], and SW620 [142], as identified by apoptotic markers or morphological characteristics. An in vivo trial with rats exposed to low doses of DON caused apoptosis in colonic tissue [15]. Autophagy was also observed as an alternative and/or complementary mode of action of DON and DON-sulfates on HT-29 cells [72], which raises the hypothesis that ADCD might be induced as a secondary RCD pathway under some circumstances. On the other hand, necroptosis is known to be initiated by perturbations of the cellular microenvironment detected by specific death receptors (e.g. FAS and TNFR) and TLRs [147], through which trichothecenes may potentially induce necroptosis, yet this hypothesis requires experimental confirmation. Because of their anti-survival nature, trichothecenes such as T-2, verrucarins, roridins, and others are getting increasing attention on their anti-cancer potential [14,137,142]. It has been reported in CECs that trichothecenes can suppress cell proliferation by inhibition of Wnt pathway or JAK/STAT pathway, and induce apoptosis via the mitochondrial pathway and Fas pathway:

1)
Cell proliferation inhibition by disrupting Wnt pathway

The canonical Wnt signaling pathway, specifically the Wnt/β-catenin/c-Myc axis, is an important mechanism by which trichothecenes regulate proliferation of CECs (Fig. 1I,J). It is known that the Wnt ligands regulate Wnt pathway through binding (Wnt-on) or not binding (Wnt-off) to the membrane-bound receptors: lipoprotein receptor-related protein (LRP) and Frizzled. The stability of cytoplasmic β-catenin, the key signal transducer of this pathway, is controlled by the destruction complex (DC). In the Wnt-off state, DC consists of AXIN as the scaffold, casein kinase 1 (CK1), glycogen synthase kinase 3 (GSK3), adenomatous polyposis coli (APC), transducing-repeat-containing E3 ubiquitin protein ligase (β-TrCP) and proteasome. β-catenin is phosphorylated by CK1 and GSK3, and subsequently ubiquitinated by β-TrCP for proteasomal degradation. In the Wnt-on state, the cytosolic protein Dishevelled is recruited to bind the intracellular regions of Frizzled and recruits DC. The associated kinases phosphorylate the cytosolic tail of LRP, providing additional binding sites for AXIN and leading to GSK3 inhibition. Together, these altered protein–protein interactions stop β-catenin proteolysis, so β-catenin can be accumulated and saturate all the binding sites in the cytoplasm and translocate into the nucleus, where it engages proteins of the T-cell-specific transcription factor/lymphoid enhancing factor (TCF/LEF) family by displacing the TCF/LEF inhibitor Groucho [148], resulting in activation of Wnt target genes such as c-Myc. The c-Myc protein is a transcription factor of many pro-proliferative genes [149]. Trichothecenes may suppress Wnt pathway by downregulating the expression of β-catenin and/or kruppel-like factor 3 (Klf3): (a) DON disrupted the β-catenin/c-Myc signaling axis by downregulating β-catenin at both mRNA and protein levels in SW480 cells, even in artificial Wnt-on state. The downregulated β-catenin suppressed c-Myc activation, thus inhibiting cell proliferation. Restoration of β-catenin or c-Myc level clearly attenuated the inhibitory effect of DON on cell proliferation [149]. Downregulation of β-catenin protein was also observed in HCT116 and SW620 cells whose proliferation was inhibited by VJ [142]. (b) Klf3 was found to be a transcription factor which binds the promotor of Wnt1 gene to promote Wnt1 expression, thus activating Wnt pathway [150]. An in vivo study found that gavaged DON suppressed the transcription of Klf3 in the intestinal tissue of mice [60], which may explain the DON-induced inhibition of Wnt pathway. However, by what mechanisms trichothecenes downregulate the levels of β-catenin and Klf3 in CECs is not concluded yet.

2)
Cell proliferation inhibition by inhibiting JAK/STAT pathway

TCN inhibits proliferation of HCT116 cells by attenuating JAK/STAT pathway. TCN treatment inactivates STAT3 by directly binding to its Src homologous region 2 (SH2) domain, independently of any upstreaming signaling alterations. Binding of TCN to the SH2 domain of STAT3 is through hydrophobic interactions and formation of hydrogen bonds with Asn538 and Trp501 located on other STAT3s in the dimerization of STAT3 through two carbonyls. This finally results in inhibition of phosphorylation, dimerization, nuclear translocation, and transcriptional activity of STAT3, which otherwise works as a transcription factor for genes involving cell survival and proliferation [29]. A recent study revealed that TCN inhibited CRC cell growth by hampering the sphingosine kinases 1 (SPHK1)/sphingosine 1-phosphate (S1P) metabolic pathway. Briefly, TCN treatment upregulated protein expression of dehydrogenase/reductase member 2 (DHRS2), which directly binds to SPHK1 mRNA to accelerate its degradation. This hampered the SPHK1/S1P metabolic pathway, resulting in inhibition of CRC cell growth [145].

3)
Apoptosis mediated by mitochondrial pathway

The mitochondrial pathway is a cysteine-aspartic acid protease (caspase or CASP)-dependent intrinsic apoptosis pathway regulated by the levels of BCL-2 family proteins [151]. A good consistency was noted between ROS generation and the viability of Caco-2 cells upon DON treatment [19], probably because DON-induced ROS generation and apoptosis share a common regulatory hub, i.e. the mitochondrion. Studies using the human colonic epithelial cell lines Caco-2 [144] and HCT116 [29,73,142,146] and colonic tissue of rats [15] demonstrated that colonic epithelial apoptosis induced by acute or chronic exposure of DON, TCN, or VJ was mediated by the mitochondrial pathway, which has been attributed to inhibition of BCL-2, an anti-apoptotic multi-domain protein of the BCL-2 family [15,29,142,144], and/or activation of BAX, a pro-apoptotic multi-domain protein of the BCL-2 family [15,29,71,73,144]. The pro-apoptotic multi-domain proteins of the BCL-2 family (e.g. BAX and BAK) are directly responsible for assembling oligomeric apoptotic pores in and permeabilizing the mitochondrial outer membrane (MOM), and their activity is positively regulated by the pro-apoptotic BH3-only proteins of the BCL-2 family (e.g. BID, BAD, BIM, NOXA, and PUMA) but negatively regulated by the anti-apoptotic multi-domain proteins of the BCL-2 family (e.g. BCL-2, BCL-xL, and MCL-1). Interactions between members of the BCL-2 family are mediated by their BH domains [151]. Several mechanisms have been found to be activated or inhibited by trichothecenes to modulate BCL-2 family protein levels and subsequently activate mitochondrial pathway (Fig. 1K):

(a)
DON can modulate BCL-2 family protein activity by activating p53 and/or CHOP via at least three possible mechanisms, e.g. RSR (see the subsection on RSR), UPR (See the section on ERS and UPR), and DDR (See the subsection on DDR), finally inducing apoptosis. It has been well established that BCL-2 family proteins are downstream signal transducers of p53, which can promote apoptosis by regulating the BCL-2 family proteins both post-translationally and transcriptionally. For example, p53 can directly activate BAK, release BAK from the MCL-1/BAK complex, inhibit BCL-2, BCL-xL, and MCL-1 by physical binding, or serve as a transcriptional factor to activate the expression of BAX, PUMA, and NOXA [107]. DON has been found to promote the hallmark events of the mitochondrial pathway in CECs. Upon inhibition of BCL-2 and activation of BAX/BAK, homo- or hetero-dimers of BAX/BAK bind to the MOM, leading to mitochondrial outer membrane permeabilization (MOMP), characterized by the opening of mitochondrial permeability transition pore (mPTP) and the loss of the mitochondrial transmembrane potential (ΔΨm) [69,71,73,146]. MOMP directly promotes the cytosolic release of apoptogenic factors (typically Cytc) which normally reside in the mitochondrial intermembrane space. The cytosolic pool of Cytc binds to apoptotic peptidase activating factor 1 (APAF1) and pro-CASP9 to form the apoptosome, which activates CASP9 [15,73,147]. The activated CASP9 catalyzes the proteolytic activation of effector caspases, i.e. CASP3 and CASP7, which are enzymes responsible for cleavage of hundreds of protein substrates to promote cell destruction [69,73,99,151]. Besides the abovementioned post-translational activation, it is interesting that p53, CASP9, and CASP3 in DON-treated Caco-2 cells were activated transcriptionally also [71,144].
(b)
The macrocyclic trichothecene VJ has been reported to modulate BCL-2 family proteins through suppressing AKT and Notch pathways to induce apoptosis in CRC cells. It is known that AKT regulates cell survival and apoptosis by phosphorylating pro-apoptotic effectors such as BCL-2 family proteins, while Notch 1 and its direct downstream target Slug, a mesenchymal progression marker, regulate cell proliferation and apoptosis by inducing BCL-2 gene expression. Excessive proliferation and epithelial to mesenchymal transition (EMT) of CRC cells are attributed to AKT hyperactivation and Notch1 upregulation. VJ can suppress the aberrant activation of the AKT and Notch1 signaling pathways to modulate BCL-2 family proteins, which activate the mitochondrial pathway, leading to the increased levels of cleaved CASP9 and CSAP3 in HCT116 and SW620 cells [142].

4)
Apoptosis mediated by Fas pathway

Fas pathway is a caspase-dependent extrinsic apoptosis pathway driven by Fas, a cell surface death receptor (DR) characterized by a Cys-rich extracellular domain and an intracellular Death Domain (DD). Fas pathway is triggered by Fas ligand (FasL), which binds to Fas and causes Fas trimerization. The Fas trimer then recruits pro-CASP8 via FAS-associated protein with death domain (FADD) as an adaptor, which has two domains: a DD and a Death Effector Domain (DED), for combination with the DD of Fas and the DED of pro-CASP8, respectively. Once recruited, Pro-CASP8 gets oligomerized and activated through self-cleavage. The activated CASP8 then activates downstream CASP3, which carries out apoptosis [147]. In the colonic tissue of rats fed DON, the protein levels of Fas, FADD, and CASP8, as the key elements of Fas pathway, were upregulated to different extents, suggesting the participation of Fas pathway in DON-induced apoptosis [15]. How DON activates the Fas pathway to induce apoptosis has not been elucidated, but it was likely to be associated with p53, which can be activated by DON via RSR (see the subsection on RSR), UPR (See the section on ERS and UPR), and DDR (See the subsection on DDR). The activated p53 functions as a transcription factor for expression of Fas/FasL to trigger the Fas pathway [107]. Activation of Fas pathway by DON was coordinated with changes in protein levels of BAX/BAK, BCL-2, Cytc, and CASP9, the key elements of the mitochondrial pathway [15]. In DON-treated Caco-2 cells, elements of both the mitochondrial and Fas pathways, e.g. p53, CASP9, CASP8, and CASP3 were transcriptionally activated [144]. These in vitro and in vivo results reveal that DON-induced apoptosis in CECs is likely a combined effect of the mitochondrial and Fas pathways (Fig. 1K).

Destruction of colonic epithelial integrity

Exposure to trichothecenes has been widely reported to impair epithelial integrity of the colon in both in vitro and in vivo studies. Acute or chronic exposure to T-2 [59,152], DON [1,3,25,26,37,44,47,110,115,116,[153], [154], [155], [156], [157], [158], [159], [160]], 3ADON, or 15ADON [117,118] disintegrated the colonic epithelium in vitro in a dose-dependent manner, as indicated by decreased transepithelial electrical resistance (TEER) and/or increased paracellular flux of markers of different sizes such as mannitol, Lucifer yellow (LY), FD-4, or fluorescein isothiocyanate-dextran (FITC-dextran) through differentiated Caco-2 cell monolayer. Animal trials confirmed that DON disrupts epithelial integrity of the colon. In rodents, DON treatment led to significant histological damages in colonic epithelium and mucosa, with reduced TEER and increased paracellular (but not transcellular) permeability of the colonic tissue [15], while gavaged FITC-dextran was transported more efficiently to serum [154]. In rabbits orally given DON, the epithelium was exfoliated to different degrees with irregular microvilli in caecum and colon, but less severely than in ileum [161]. 15ADON caused higher permeability than DON and 3ADON on Caco-2 cell monolayer [118]. Destruction of the intestinal epithelial integrity by trichothecenes may results in various intestinal disorders. It may facilitate the initiation and progression of inflammatory bowel and celiac disease [154], and increase colonization of enteric pathogens (e.g. E. coli and Campylobacter jejuni) and their translocation to liver and spleen [47,157,162,163]. In contrast, DON-3-Glu, DOM-1 and 3-epi-DON did not exhibit significant negative effects on intestinal epithelial integrity [37,43,117,163].

Trichothecenes impair the colonic epithelial integrity by reducing the synthesis of cell junction proteins, which are essential for the assembly of functional cell junctions. Expression of TJ constituents claudins and occludin, and/or the scaffold protein ZO-1 were downregulated in Caco-2 cells exposed to T-2 [59,152] or DON [25,109,115,[155], [156], [157],159], and also in mouse rectal epithelial cells CMT93-II exposed to DON [164]. In vivo trials confirmed that expression of TJ constituents could be downregulated by DON at both mRNA and protein levels. The mRNA transcription of occludin and ZO-1 in the intestine tissue of mice gavaged with DON were downregulated [60]. Another trial with rats revealed that dietary DON suppressed the protein expression of occludin, claudins, and ZO-1 in the colonic epithelium [15]. Occludin expression was also downregulated by DON at NOAEL in mice with experimentally induced colitis [121]. By reducing expression of TJ constituent and scaffold proteins, trichothecenes inhibit the assembly of TJs, and disturb the continuity and distribution of TJ strands in the cells, resulting in increased paracellular permeability of the epithelium [115]. DON treatment can also inhibit the assembly of AJs by downregulating the expression of AJ constituent protein E-cadherin [3,155] and the linker protein β-catenin [149] in different colonic epithelial cell lines, leading to disintegration of the epithelium.

How trichothecenes regulate the levels of cell junction proteins is not clearly understood, but probably by multiple mechanisms. It has been suggested that DON can suppress expression of TJ and AJ proteins by regulating MAPK, Wnt, and/or JAK/STAT pathways, and inhibit assembly of TJs by interrupting protein kinase A (PKA) pathway (Fig. 1L,M):

1)
Regulation of cell junction protein levels by MAPK, Wnt and JAK/STAT pathways

Early studies showed that activation of ERK MAPK pathway, as consequence of DON exposure, was involved at least partially in the suppression of claudin expression. Using ERK-specific inhibitor in DON-treated CECs was found to restore claudin-4 protein expression and epithelial barrier function [165,166]. Activation of ERK MAPK by DON also suppressed the expression of cadherin genes in the Caco-2 cell monolayer [3]. How MAPKs mediate the downregulation of these cell junction proteins in CECs has not been well elucidated, but presumably via zinc-finger transcription repressors, i.e. the Snail family proteins Snail and Slug. Snail and Slug are known to be transcriptionally activated by MAPKs (especially ERK1/2 and JNK) through the transcription factor AP-1 [[167], [168], [169]], and repress the expression of TJ and AJ proteins in transcriptional and/or posttranscriptional manners [[169], [170], [171], [172], [173], [174], [175]]. A study by Nakayama et al. [164], however, showed that inhibition, instead of activation, of ERK MAPK impaired TJ protein expression, while Van De Walle et al. [25] suggested that the reduced expression of TJ protein does not result from activation or inhibition of the NF-κB, ERK, or JNK inflammatory cascades. This suggests that trichothecene-induced reduction of TJ protein expression is likely mediated by multiple mechanisms. Disruption of Wnt pathway was found to be another involved mechanism. The transcriptional activity of TCF/LEF can be activated by β-catenin through Wnt pathway. The activated TCF/LEF then promotes claudin-1 gene transcription by binding its promoter [176]. DON can disrupt Wnt/β-catenin signaling by downregulating the expression of β-catenin and Klf3 (See the subsection on regulation of CEC viability), which likely suppresses the transcriptional activity of TCF/LEF and transcription of claudin-1 gene. Activation of JAK/STAT pathway by DON was also found to participate in the downregulation of occludin expression [121]. Unfortunately, the mechanism by which JAK/STAT regulates occludin expression has not been well described.

2)
Regulation of TJ assembly by inhibiting PKA

PKA and its downstream target vasodilator-stimulated phosphoprotein (VASP), an actin-binding protein, are believed to be involved in DON-induced disruption of TJ assembly. In unstressed cells, PKA can phosphorylate VASP, thereby disrupting its ability to bundle actin of the cytoskeleton and promoting its relocation to tight junctions [177], where the phosphorylated VASP promotes the assembly of the TJs through the binding of claudin and occludin with cytoplasmic scaffold protein ZO-1. It was found in Caco-2 cells that DON inhibited PKA by reducing the level of the catalytic subunit of PKA, which inhibited phosphorylation of VASP and assembly of TJs [3] (Fig. 1M).

Regulation of mucus composition

Acute or chronic exposure to trichothecenes affects the colonic epithelial mucus by altering the production of mucus components by CECs. Protein production and mRNA transcription of both the membrane-bound mucins (MUC1, MUC3) and the secretory mucin MUC2 of human goblet cells (HT29-16E cells) were decreased by DON exposure in a time- and dose-dependent manner (0.1–100 μM), while neither cell viability, integrity, morphology nor the number of secretory granules was affected by DON at ≤10 μM. Composition modification of secretory granules by DON was also observed by transmission electron microscopy (TEM) [48]. In vivo trial with mice treated with DON at NOAEL confirmed the ability of DON to decrease MUC2 expression in colon [121]. In contrast, a study by Wan et al. [119] exhibited that DON dose-dependently upregulated the transcription of secretory mucins (MUC5AC, MUC5B) and membrane-bound mucins (MUC1, MUC4, and MUC17), but downregulated that of MUC3 in Caco-2 cells. Exposure to DON lowered the production of acidic and neutral mucins, as visualized by Alcian blue/periodic acid-schiff staining, but elevated the total mucin-like glycoprotein production, as suggested by enzyme-linked lectin assay (ELLA) [119]. Though less studied than DON, T-2 was also found to inhibit the production of MUC2 by Caco-2 cells, HT-29, and HT29-H cells treated at a dose of 1/4 IC50 for 24 h. The following in vivo trial using mice confirmed that T-2 treatment decreased MUC2 production in the colon [59]. Exposure to different combinations of DON and NIV, however, resulted in irregular modulation of MUC5AC and MUC5B mRNA expression and secretion, as well as total mucin-like glycoprotein secretion in cocultures of Caco-2 (absorptive-type) and HT29-MTX (secretory-type) cells at varying ratios [178], implying that mixed trichothecenes modulate mucin production in cocultures of CECs by complex mechanisms. Other mucus components may also be regulated by trichothecenes. Even nanomolar doses of DON inhibited the secretion of TFFs by HT29-16E cells, which could prevent wound healing of Caco-2 cell monolayer [4]. These results implies that DON and other trichothecenes at low doses found in foodstuffs may alter the expression and production of mucus components by human CECs. Overall, under the influence of DON or T-2, downregulation of mucus components appears to be the predominant trend. However, occasional upregulation may occur in some contexts, suggesting that trichothecenes regulate the expression of mucus components via complex mechanisms which require further investigation.

High doses of trichothecenes can impair mucin production by reducing the number of goblet cells or secretory granules therein, while subtoxic doses of trichothecenes can reduce mucin production through modulation of signal pathways and gene expression in goblet cells without affecting the cell viability, integrity, or morphology [48,179]. MAPKs and IRE1β have been reported to mediate these alterations:

1)
Inhibition of mucin and TFF production by PKR/MAPK and JAK/STAT pathways

DON reduced production of mucins (MUCs 1, 2 and 3) in HT29-16E cells by inhibiting the expression of RELM-β [48]. RELM-β is known as a mucosecretagogue, which promotes the expression and secretion of MUC2 and MUC5AC in HT29‑16E cells [9]. DON activated PKR and p38 MAPK (See the subsection on RSR) to inhibit the expression of RELM-β. Inhibition of mucin expression resulted in depletion of mucin from the granules. Applications of PKR and p38 MAPK inhibitors prevented the inhibitory effect of DON on RELM-β mRNA expression, and restored the production of mucins. In contrast, inhibitors of NF-ĸB or ERK1/2 failed to counteract the inhibitory effects of DON on RELM-β and mucin expression, suggesting that NF-ĸB and ERK1/2 MAPK did not mediate the DON-induced inhibition of mucin production [48]. Inhibition of the expression of TFFs by DON in HT29-16E cells depended on the activation of PKR and the MAPKs p38 and ERK1/2. Application of specific inhibitors against PKR, p38 MAPK or ERK1/2 MAPK counteracted the inhibitory action of DON on TFFs mRNA expression [4]. Activation of JAK/STAT pathway by DON was also found to participate in the downregulation of MUC2 expression, but the underlying mechanisms were not well described [121] (Fig. 1N).

2)
Inhibition of mucin production by suppressing IRE1β

In contrast to DON, T-2 inhibits mucin production presumably by suppressing IRE1β, instead of by activating the p38, JNK MAPKs or NF-κB signaling pathways. IRE1β, as a homologue of IRE1α, is not only an ERS sensor, but also considered a positive regulator of mucin expression [59]. Despite obscure mechanisms, expression levels of IRE1β and MUC2 have been noticed to be positively correlated in human colon mucosa [180], mouse colonic epithelium [181], and respiratory epithelial cells [182]. T-2 exposure caused concomitant decrease in IRE1β and MUC2 production in human colonic epithelial cell lines and in mouse colonic tissue [59]. IRE1β is responsible for ER homeostasis [183] and microbiota-induced maturation [184] of goblet cells, and probably involved in expression of genes linked to mucin production, such as AGR2, CLCA1/3, SPDEF, and mucin glycosylating enzymes [182]. It is presumed that T-2 inhibits MUC2 production by interrupting the normal functions of IRE1β on goblet cells, but the exact mechanisms by which T-2 induces decrease of MUC2 production via IRE1β remains unclear and yet to be studied.

Conclusions and future research directions

Conclusions

Toxic effects of trichothecenes on CECs are highly dose-dependent. A slight stimulation can increase cell viability (hormesis), a mild stimulation can inhibit cell proliferation and induce inflammation, a moderate stimulation can lead to programed cell death (e.g. apoptosis), and a potent stimulation can disable the cells from self-regulation and kills the cells through necrosis. While CECs share a lot in common with other cell types in response to trichothecene exposure, several key distinctions have been noticed: (1) Due to the high sensitivity of proliferating cells to trichothecenes, the fast renewal of the epithelium makes CECs highly sensitive to trichothecenes. (2) Contrary to hematopoietic lineages, HCK does not participate in RSR in CECs. (3) The colonic epithelium maintains it integrity by cell junctions to function properly, but the cell junctions are highly susceptible to destruction by trichothecenes. (4) The abundance of RELM-β in the thick colonic epithelial mucus layer makes CECs particularly vulnerable to regulation by trichothecenes in terms of mucin production and secretion.

The ribosome, the mitochondrion, and the nucleus of CECs are probably the primary or early targets with which trichothecenes may directly interact, leading to the respective primary toxic effects: translation inhibition, ROS overproduction, and DNA damage. These primary toxic effects further induce ERS, oxidative stress, inflammation, inhibition of cell proliferation, apoptosis, destruction of epithelial integrity, and alteration of mucin production. These toxic effects are mediated by a complex network of signaling pathways, including MAPK pathway, PERK/eIF2/ATF4 pathway, Ire1α/XBP1 pathway, NF-κB pathway, p53 pathway, JAK/STAT pathway, Wnt pathway, mitochondrial pathway, and Fas pathway, etc., with extensive crosstalk between different pathways. One signaling pathway can mediate multiple toxic effects, and a single toxic effect can be regulated by multiple signaling pathways. The combined effects of multiple signaling pathways can be additive, synergistic, or antagonistic. The complex signaling network involved in trichothecene toxicity makes it a challenge to clearly elucidate the underlying molecular mechanisms. A great effort has been made in this review to characterize possible dominant mechanisms of trichothecene toxicity, as well as possible relationships between different toxic effects. This may reveal novel preventative/therapeutic strategies for trichothecene toxicosis.

Current research on trichothecene toxicity on CECs depends largely on in vitro studies using colon-derived epithelial cell lines (e.g. Caco-2, HT-29, HCT116, SW480, SW620, SW742, COLO201, and HCEC-1CT), which have been widely used as in vitro models in toxicological studies, especially in revealing molecular mechanisms. However, due to lack of systemic complexity, absence of metabolic processing, oversimplified cellular microenvironment, absence of dynamic microenvironments, and short exposure duration, in vitro studies may limit the understanding on the toxic effects in physiological environments and thus reduce translational relevance. A number of in vivo studies have been performed for investigation of trichothecene toxicity on CECs. Though the complexity of the physiological environment in animal intestine poses challenges for precisely studying the effects of individual toxins on CECs, the results from these in vivo studies were generally consistent with those from the in vitro studies, with no notable discrepancies observed.

Future research directions

For a clearer understanding on the mechanisms of trichothecene toxicity on CECs, some key topics deserve further study:

1)
The ultimate relationship between cytotoxicity and trichothecene-ribosome interaction supports the suggestion that ribosome is a primary target of trichothecenes, but the current knowledge on structure–toxicity relationship of trichothecene molecules have been based on yeast ribosome. Future study should establish such relationships using human or, at least mammalian ribosomes. While the role of PKR as a mediator of trichothecene-induced RSR in CECs has been substantiated, the role of ZAKα in this process is a deduction based on the studies in other cell types and thus needs to be experimentally verified in future study. Additionally, the mechanistic basis for the preferential activation of PKR vs ZAKα in CECs following trichothecene exposure remains an unresolved question.
2)
How trichothecenes induce ERS is not clearly understood. It is logical to believe that inhibition of protein synthesis by trichothecenes leads to accumulation of truncated peptides in the ER, thus causing ERS followed by UPR, but direct evidences in CECs are still lacking. While the ERS sensors PERK and IRE1α have been found to be activated by trichothecenes in CECs to initiate UPR through their respective signaling pathways, whether ATF6, a third ERS sensor, is involved in trichothecene-induced UPR signaling is an interesting question to answer.
3)
While trichothecenes can be absorbed into the mitochondria, their direct interactions with the mitochondria (particularly with the ETC), their potential ability to promote electron leak, and the relationship between cytotoxicity and trichothecene-mitochondrion interaction remain to be evidenced and characterized. What is more, whether other potential sources of ROS such as peroxisomes, NADPH oxidase, lipoxygenase, cyclooxygenases, and cytochrome P450s play any roles in trichothecene-induced oxidative stress is worth further investigation.
4)
While trichothecenes can be absorbed into the nucleus, their direct interactions with the nucleus (particularly with the DNA), and the influence of their molecular structures on these interactions remain to be evidenced and characterized. Whether and how trichothecenes really induce DNA damage by direct interaction with DNA in CECs are yet to be directly evidenced. Activation of ATM/p53 signaling as trichothecene-induced DDR in CECs needs further characterization.
5)
Inflammation has been shown to be a secondary response following trichothecene-induced RSR, ERS, UPR, and/or oxidative stress, mediated by MAPK, NF-κB, JAK/STAT, PERK/eIF2/ATF4, and IRE1α/XBP1 pathways and their feedbacks. Whether the combined effects of these signaling pathways on inflammation are additive, synergistic, antagonistic, or even independent is crucial for a better understanding on the mechanism of the pro-inflammatory effect of trichothecenes.
6)
Trichothecenes have been well documented to suppress cell proliferation and induce apoptosis in CECs. β-catenin, as a key signal transducer in Wnt pathway and a linker protein between AJs and the cytoskeleton, seems to be a critical target for trichothecenes to regulate cell proliferation and colonic epithelial integrity, but how trichothecenes regulate the activity and level of β-catenin remains to be elucidated. Although p53 and CHOP have been well documented to induce apoptosis by regulating BCL-2 family proteins and FAS/FASL, direct evidence is still needed in trichothecene-exposed CECs. The anti-survival nature of trichothecenes may make them promising candidates as anti-cancer agents for gastrointestinal and other cancers. A broad screening followed by thorough investigation is necessary on their efficacy and safety.
7)
Although trichothecenes have been documented to destruct the colonic epithelial integrity by regulating MAPK, Wnt, JAK/STAT, and PKA pathways, and impair the colonic epithelial mucus layer by regulating MAPK and JAK/STAT pathways and suppressing IRE1β, these mechanisms are still obscure, with more details yet to be further elucidated.
8)
Since the colonic microbiota interact intimately with the colonic mucus and epithelium, any trichothecene-induced dysbiosis may disrupt this interaction, contributing to gut toxicity. Therefore, future investigation on potential influence of trichothecenes on the colonic microbiota is expected to clarify the role of gut microbes in trichothecene toxicity and may reveal novel microbiota-targeted therapeutic strategies.
9)
Unveiling the toxicological mechanisms of trichothecenes is expected to reveal potential therapeutic or preventative strategies to mitigate trichothecene toxicity in the gut. For example, antioxidants may counteract oxidative stress, MAPK and/or NF-κB inhibitors could reduce inflammation, and agents targeting MAPK, Wnt, and/or JAK-STAT pathways may preserve epithelial integrity, etc. Potential therapeutic strategies based on toxicological mechanisms of trichothecenes need further exploration.
10)
Although a number of in vivo studies confirmed the findings of in vitro studies, in vivo data, especially from chronic exposure trials, remain insufficient and require further investigation. Moreover, translating in vitro (cell models) and in vivo (animal models) findings to human remains challenging due to differences in toxicokinetics and physiology. These gaps highlight the need for developing more physiologically relevant models.

Compliance with ethics requirements

This article does not contain any studies with human or animal subjects.

CRediT authorship contribution statement

Shao-Ji Li: Conceptualization, Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing. Chun-Min Yang: Conceptualization, Funding acquisition, Supervision, Writing – review & editing. Shiyi Ou: Funding acquisition, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank the editor and reviewers for comments. This study was funded by Key Scientific Research Platforms and Projects for Universities in Guangdong Province (2024GCZX011), and Guangdong Provincial Key Discipline Construction Program for Enhancing Research Capacity (2024ZDJS089).

Biographies

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Shao-Ji Li, Associate Professor and Engineer, earned his Bachelor’s degree in biological engineering from Northeastern University (China), Master’s degree in biological engineering from Inha University (South Korea), and PhD in veterinary science from Ghent University (Belgium). He has held various research positions, including Laboratory Technician at Gil Medical Center (affiliated with Gachon University, South Korea), Assistant Researcher at the Korea Centers for Disease control and Prevention (CDC), Postdoctoral Researcher at the University of Tennessee Health Science Center (USA), and Technical Director at SignalDT Inc. (China). Currently he works at the Department of Food Quality and Safety, School of Engineering, Guangzhou College of Technology and Business, where he teaches the course Food Toxicology and conducts research mainly on mycotoxins, food toxicology, and nucleic acid detection technology. He has participated in research projects funded by the Rural Development Administration (RDA) of South Korea and the U.S. National Institutes of Health (NIH). His contribution to this article includes: conceptualization, data curation, investigation, methodology, writing- original draft, writing-review & editing.

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Chun-Min Yang, Professor, Director of Department of Food Quality and Safety, School of Engineering, Guangzhou College of Technology and Business. She teaches courses such as Food Microbiology and Food Microbiological Testing, and her research focuses on food fermentation, and safety and control of microbial metabolites. In recent years, she has led or participated in 16 scientific research projects funded by the provincial departments and the college. Additionally, she has successfully completed an enterprise research project with funding of 300,000 RMB. She has also supervised multiple students in innovation and entrepreneurship projects that received provincial grants, including Guangdong’s Youth League’s Climbing Plan for College Student Innovation and provincial College Student Innovation and Entrepreneurship Training Programs. Some of these projects were recommended for national recognition. Recently she has published over 20 research articles, including 8 as the first or corresponding author, among which 2 are indexed in Peking University’s Chinese Core Journals and 2 are SCI-indexed. Her contribution to this article includes: funding acquisition, methodology, writing-review & editing.

graphic file with name fx3.jpg

Shiyi Ou, Professor, Master's and Doctoral Supervisor, holds a PhD in Food Science from South China University of Technology. He completed postdoctoral research in the Department of Applied Biology and Chemical Technology at The Hong Kong Polytechnic University. His primary research focuses on the safety and control of food processing. He has presided over 20 provincial/ministerial projects and 4 NSFC projects, received 3 provincial/ministerial awards, and holds 15 authorized invention patents (3 of which have been transferred). He has published more than 150 SCI-indexed articles, which have been cited over 6,600 times (H-index: 43), and authored 2 academic monographs. Currently, he serves as the Deputy Dean of the School of Engineering at Guangzhou College of Technology and Business, Council Member of the Chinese Institute of Food Science and Technology, Committee Member of the Guangdong Food Safety Commission, Council Member of the Guangdong Light Industry Association, and Editorial Board Member for journals such as Journal of Chinese Institute of Food Science and Technology, Science and Technology of Food Industry, Modern Food Science & Technology, Journal of Food Safety and Quality, and Foods. His contribution to this article includes: funding acquisition, methodology, writing-review & editing.

Contributor Information

Chun-Min Yang, Email: hbycm@gzgs.edu.cn.

Shiyi Ou, Email: oushyi@gzgs.edu.cn.

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PERMALINK

Impacts of trichothecene mycotoxins on human colonic epithelial cells: molecular mechanisms and signaling pathways

Shao-Ji Li

Chun-Min Yang

Shiyi Ou

Graphical abstract

Highlights

Abstract

Background

Aim of review

Key scientific concepts of review

Introduction

Introduction of colonic epithelium

Introduction of trichothecene mycotoxins

Table 1.

Outline and aim of this review

Inhibition of protein synthesis and initiation of ribotoxic stress response

Inhibition of protein synthesis

Fig. 1.

Initiation of ribotoxic stress response

Induction of endoplasmic reticulum stress (ERS) and unfolded protein response (UPR)

Induction of oxidative stress

Observations of trichothecene-induced oxidative stress

Mechanism of trichothecene-induced ROS production

Table 2.

Activation of NF-κB by ROS

Mechanism of trichothecene-induced RNS production

Induction of DNA damage and DNA damage response

Induction of DNA damage

Induction of DNA damage response

Modulation of inflammatory response

Modulation of inflammatory markers

Mechanisms of modulation of inflammatory response

Regulation of CEC viability

Influence of trichothecenes on CEC viability

Mechanisms of regulation of CEC viability

Destruction of colonic epithelial integrity

Regulation of mucus composition

Conclusions and future research directions

Conclusions

Future research directions

Compliance with ethics requirements

CRediT authorship contribution statement

Declaration of competing interest

Acknowledgments

Biographies

Contributor Information

References

ACTIONS

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