Mechanism of deoxynivalenol-induced neurotoxicity in weaned piglets is linked to lipid peroxidation, dampened neurotransmitter levels, and interference with calcium signaling
Graphical abstract
Introduction
Mycotoxins, produced by various fungi, are toxic secondary metabolites that have similar biological activities (Ren et al., 2015; Wang et al., 2016). Deoxynivalenol (DON) is a type B trichothecene mycotoxin produced mainly by Fusarium graminearum and Fusarium culmorum and is commonly found worldwide (Wang et al., 2018a, 2019a). For the past half century, ingesting deoxynivalenol-contaminated grain has been associated with large outbreaks of noninfectious gastroenteritis across Europe and India (Pestka, 2010; Brera et al., 2013; Kimanya et al., 2014), making the mycotoxin a serious global food-security issue. Some studies have suggested that DON exposure induces feed refusal, decreases animal productivity, damages organs, increases disease incidence, and causes nutrient malabsorption (Li et al., 2015). Over 40 countries have introduced regulations to control DON levels in food and feed.
Response to DON exhibits clear interspecific differences. Pigs are among the most sensitive, followed by rodents, dogs, cats, poultry, and ruminants (Prelusky, 1994; Wang et al., 2019b). Deoxynivalenol-associated toxicity may be due to the compound's ability to induce free radical production that then damages cell membranes and DNA by causing oxidative stress (Wu et al., 2014). The brain is particularly susceptible to oxidative damage because it contains high levels of unsaturated fatty acids, catecholamine, and oxidative capability. Multiple experiments have demonstrated that DON triggers lipid peroxidation, as evidenced by increases in reactive oxygen species and decreases in antioxidant-enzyme activity (Strasser et al., 2013; Daotong et al., 2014). Liver is the main organ of lipid metabolism, which is closely related to oxidative stress, DON can cause hepatotoxicity damage (Mikami et al., 2010). Previous study has shown that a subchronicexposure to low doses of DON causes changes in the normal liver metabolism of xenobiotics (Gouze et al., 2006), however, the effect of DON on lipid peroxidation in brain has not been reported in detail.
In addition to its oxidative effects, DON can influence neurotransmitter secretion in the brain in vivo. In particular, altering the levels of 5-hydroxytryptamine (5-HT, serotonin) is the mechanism underlying well-documented emetic effects in pigs (Vesonder et al., 1973; Endo et al., 2000; Wu et al., 2013). In addition to 5-HT, studies have also identified deoxynivalenol-related increases of norepinephrine, coupled with a decrease in dopamine among piglets (Prelusky et al., 1992; Prelusky, 1993).
Calcium ions control diverse cellular processes, including muscle contraction, neurotransmitter release, gene expression, and cell proliferation (Tsien and Tsien, 1990). Thus, Ca2+ homeostasis is crucial for normal physiological and biochemical processes in cells. Disruption of Ca2+ homeostasis could cause reduced or abnormal generation of functional neuronal circuits in the maturing hippocampus (Helmuth et al., 2005). In support of the link between Ca2+ and DON, we previously discovered that DON treatment in chicks significantly reduced calcium-binding signal-transducer calmodulin mRNA and protein expression (Wang et al., 2018a).
These findings suggest that the mechanism underlying DON toxicity would involve Ca2+/calmodulin-dependent kinase II (CaMKII), a major component of excitatory synaptic factors. CaMKII has been linked to regulation of virulence-factor-induced apoptosis (Liu and Templeton, 2010; Teng et al., 2017; Julieta et al., 2009). However, very few studies have investigated whether DON deregulates calcium homeostasis through the Ca2+/CaM/CaMKII pathway in pigs. Thus, in this study, we investigated the effects of DON on lipid peroxidation, neurotransmitters, and calcium levels in piglet brain tissue. Our findings should improve current understanding of the mechanisms underlying DON neurotoxicity.
Section snippets
Reagents and kits
All chemicals were analytical grade. DON (CAS no. D0156-5 MG) was purchased from Sigma (St. Louis, MO). SOD, GSH-Px, NO, MDA, 5-HT, NE, DA, and GABA ELISA kits were obtained from Yuanye Biotechnology (Shanghai, China). The Ca2+ assay kit was purchased from Leadman Biochemical (Beijing, China). Trizol reagent was purchased from Invitrogen Biotechnology (Shanghai, China). StarScript II First-strand cDNA Synthesis Mix was purchased from GenStar Biotechnology (Beijing, China). Bestar® SybrGreen
DON alters hippocampal morphology
Control hippocampus cells exhibited complete nuclei and subcellular organelles (Fig. 1, A1), including normal mitochondrial structure and threadlike cristae (Fig. 1, A2). In contrast, under 1.3 mg/kg DON treatment, we observed absent nuclei, severe cytoplasmic swelling, as well as chromatin aggregation and condensation (Fig. 1, B1). Moreover, hippocampal cells displayed clear vacuolization (Fig. 1, B2). At 2.2 mg/kg DON, nuclear and subcellular organelles were absent. We also observed
Discussion
Previous study have shown that DON can induce toxic effects and apoptosis in piglet hippocampal nerve cells via the MAPK signaling pathway in vitro (Wang et al., 2018b). However, in this study, we demonstrated that 1.3 mg/kg and 2.2 mg/kg DON successfully induced damage to brain cells of weaned piglets in vivo, evident through observations of nuclear shrinkage and fragmentation, chromatin aggregation, mitochondrial swelling, as well as vacuolization.
Oxidative stress (cellular damage from
CRediT authorship contribution statement
Xichun Wang: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Visualization, Writing - original draft, Writing - review & editing. Xiaofang Chen: Data curation, Formal analysis, Investigation, Resources, Validation, Visualization, Writing - review & editing. Li Cao: Data curation, Formal analysis, Resources, Writing - review & editing. Lei Zhu: Data curation, Resources. Yafei Zhang: Data curation, Resources. Xiaoyan Chu: Data curation,
Declaration of competing interest
The authors declare that they have no conflict of interest.
Acknowledgment
This study was supported by the National Natural Science Foundation of China [grant No. 31472250] and the Project of Modern Agricultural Industry and Technology System of Anhui Province [grant No. AHCYJSTX-05-07]. We wish to thank anonymous reviewers for their kind advice.
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