No-observed adverse effect levels of deoxynivalenol and aflatoxin B1 in combination induced immune inhibition and apoptosis in vivo and in vitro.

Mycotoxins are toxic metabolites produced by fungal species, commonly exist in animal feeds, and pose a serious risk to human as well as animal health. But limited studies have focused on combined effects of no-observed adverse effect levels. In vivo study, 6 weeks old twenty-four mice were individually exposed to Deoxynivalenol (DON) at 0.1 mg/kg BW, Aflatoxin B1 (AFB1) at 0.01 mg/kg BW, and mixture of DON and AFB1 (0.1 mg/kg BW and 0.01 mg/kg BW, respectively) for 28 days. Then, DON at 0.5 µg/mL, AFB1 at 0.04 µg/mL, and mixtures of DON and AFB1 (0.5 µg/mL, 0.04 µg/mL, respectively) were applied to porcine alveolar macrophages (PAMs) in vitro study. Our in vivo results revealed that the combined no-observed adverse effect levels of DON and AFB1 administration decreased IgA and IgG levels in the serum, the splenic TNF-α, IFN-γ, IL-2 and IL-6 mRNA expression and T-lymphocyte subset levels (CD4+ and CD8+) in the spleen. Additionally, the combined administration increased caspase-3, caspase-9, Bax, Cyt-c, and decreased Bcl-2 protein expression. Taken together, the combined no-observed adverse effect levels of DON and AFB1 could induce immunosuppression, which may be related to apoptosis. This study provides new insights into the combined immune toxicity (DON and AFB1).

Nontoxic Concentration of Ochratoxin A Aggravates Renal Fibrosis Induced by Adriamycin/Cyclosporine A Nephropathy via TGF-ß1/SMAD2/3.

Ochratoxin A (OTA) is the most common contaminant in food and feed, which causes nephrotoxicity. Studies revealed that a low level of OTA contamination could also cause physiological dysfunction. Chronic kidney disease (CKD) has become an important public health problem with increasing morbidity. However, the potential effect of nontoxic OTA on CKD remains uncertain. In this study, adriamycin (ADR) and cyclosporine A (CSA) were used to stimulate glomerular nephropathy and tubular nephropathy, respectively. Renal injury was aggravated due to OTA (0.25 mg/kg) exposure in the mouse nephropathy models, assessing by renal histomorphology and the detection of blood urea nitrogen (BUN) and serum creatine (SCr) levels. We noticed that nontoxic dosage of OTA increased the expression of fibrotic factors, α-smooth muscle actin (α-SMA), and Vimentin in a nephropathic mouse, which indicated the exacerbation of ADR/CSA-induced renal fibrosis. We conducted in vitro experiments in glomerular mesangial cells and renal tubular epithelial cells. Nontoxic concentration of OTA was found to exacerbate the cytotoxicity of ADR/CSA and intensify renal fibrosis by activating TGF-ß1/SMAD2/3. Thus, this study may provide convincing evidence for the prevention of CKD aggravation and the renewal of food hygiene standards in mycotoxin contamination.

Ochratoxin A induced differentiation nephrotoxicity in renal tubule and glomeruli via autophagy differential regulation.

Ochratoxin A (OTA) is a mycotoxin that mainly causes nephrotoxicity. The single nephrotoxicity of OTA exposure on glomeruli or renal tubule had been well documented, however, the comparison toxicity between it is still unclear. Here, C57BL/6 mice and two types of nephrocyte were treated with concentration-gradient OTA to explore its differentiation nephrotoxicity. Results showed that OTA induced nephrotoxicity in vivo and in vitro, manifested as the deteriorative kidney function in mice and the cut-down cell viability in nephrocyte. Besides, results of murine kidney pathological section and IC50 of two types nephrocyte indicated that OTA-induced toxicity in renal tubule was higher than its in glomeruli. In addition, OTA exposure induced autophagy signaling differentiation expression. It revealed that autophagy was implicated in OTA-induced differential nephrotoxicity in glomeruli and renal tubule. Altogether, we proved that OTA induces a differentiation nephrotoxicity in glomeruli and renal tubule, and it is related to autophagy differential regulation.

Phenethyl isothiocyanate as an anti-nutritional factor attenuates deoxynivalenol-induced IPEC-J2 cell injury through inhibiting ROS-mediated autophagy.

Deoxynivalenol (DON) is considered to be the most harmful mycotoxin that affects the intestinal health of animals and humans. Phenethyl isothiocyanate (PEITC) in feedstuff is an anti-nutritional factor and impairs nutrient digestion and absorption in the animal intestinal. In the current study, we aimed to explore the effects of PEITC on DON-induced apoptosis, intestinal tight junction disorder, and its potential molecular mechanism in the porcine jejunum epithelial cell line (IPEC-J2). Our results indicated that PEITC treatment markedly alleviated DON-induced cytotoxicity, decreasing the apoptotic cell percentage and pro-apoptotic mRNA/protein levels, and increasing zonula occludens-1 (ZO-1), occludin and claudin-1 mRNA/protein expression. Meanwhile, PEITC treatment ameliorated DON-induced an increase of the inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) mRNA levels and intracellular reactive oxygen species (ROS) level, and a decrease of glutathione peroxidase 1 (GPx1), superoxide dismutase 2 (SOD2), catalase (CAT) and heme oxygenase 1 (HO-1) mRNA levels. Additionally, PEITC treatment significantly down-regulated autophagy-related protein 5 (ATG5), beclin-1 and microtubule-associated protein 1 light chain 3B (LC3-Ⅱ) mRNA/protein levels, decreased the number of green fluorescent protein-microtubule-associated protein 1 light-chain 3 (GFP-LC3) puncta and phosphatidylinositol 3 kinase (PI3K) protein expression, and up-regulated phospho-protein kinase B (p-Akt) and phospho-mammalian target of rapamycin (p-mTOR) protein expression against DON. However, the activation of autophagy by rapamycin, an autophagy agonist, abolished the protective effects of PEITC against DON-induced cytotoxicity, apoptosis and intestinal tight junction disorder. Collectively, PEITC could confer protection against DON-induced porcine intestinal epithelial cell injury by suppressing ROS-mediated autophagy.

Combined protective effects of icariin and selenomethionine on novel chronic tubulointerstitial nephropathy models in vivo and in vitro.

Chronic tubulointerstitial nephropathy (CTIN) is one of the most common kidney diseases. However, treatment for CTIN has multiple limits. Adjuvant therapy through nutritional regulation has become a hot research topic at present. Icariin (ICA), an extraction of Chinese herbal medicine epimedium, has many pharmacological functions including anti-inflammation and tonifying kidney. Selenomethionine (SeMet) possesses the effects of antioxidant and lightening nephrotoxicity. However, little is known about the combined nephroprotection of them. This study was investigated to evaluate the joint effects of ICA and SeMet on CTIN and explore the mechanism. Based on a novel CTIN model developed in our previous study, mice were randomly divided into five groups (a: control; b: model; c: model + ICA; d: model + SeMet; e: model + ICA + SeMet). Renal tubule epithelial cells were treated with cyclosporine A and ochratoxin A without/with ICA or/and SeMet. The results showed that ICA or/and SeMet ameliorated CTIN by inhibiting the uptrends of blood urine nitrogen, serum creatinine, urine protein, urine gravity, histopathological damage degree and collagen I deposition. ICA or/and SeMet also increased cell proliferation and decreased apoptosis and the expression of transforming growth factor-beta 1 and α-smooth muscle actin. Emphatically, ICA and SeMet joint had better nephroprotection than alone in most indexes including fibrosis. Furthermore, ICA and SeMet joint decreased the activation of toll-like receptor 4 (TLR4)/NFκB pathway induced by CTIN. TLR4 overexpression counteracted the joint protection of ICA and SeMet. Therefore, ICA and SeMet in combination could protect against CTIN through blocking TLR4/NFκB pathway. The study will provide novel insights to explore an adjuvant therapeutic orientation.

Combination of zinc and selenium alleviates ochratoxin A-induced fibrosis via blocking ROS-dependent autophagy in HK-2 cells.

BACKGROUND: Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium. The key target organ of OTA toxicity is the kidney, which has a significant impact on human health. Recently, nutrition regulation is suggested to be an effective protection against mycotoxins contamination. The current study investigated the combined protective effects of zinc and selenomethionine (SeMet) (a major component of organic selenium) on OTA-induced renal fibrosis and their potential mechanisms in human renal proximal tubule epithelial cells (HK-2 cells). METHODS: Cytotoxicity of different concentrations of OTA, zinc and SeMet on HK-2 cells was detected by cell viability, lactate dehydrogenase (LDH) and apoptotic nuclei assays. The expression of fibrosis biomarkers was detected by Real-Time PCR, western blotting and indirect immunofluorescence assays. The production of reactive oxygen species (ROS) was detected by ROS assay kit. The protein expression of autophagy biomarkers was detected by western blotting assay. RESULTS: Cytotoxicity was induced by OTA treatment in a dose-dependent manner, and it was attenuated by zinc or SeMet application in HK-2 cells. Zinc or SeMet application also down-regulated the expression of fibrosis biomarkers, and the combination of them displayed better effects. In addition, OTA increased intracellular ROS level and activated autophagy in a dose-dependent manner, and it was reversed by zinc and SeMet combined application. With the treatment of hydrogen peroxide (H2O2) or rapamycin (the specific activator of autophagy), the combined protective effects of zinc and SeMet were abolished. CONCLUSIONS: Zinc and SeMet application alleviated OTA-induced cytotoxicity and fibrosis in HK-2 cells. Combination of them was more effective than its individual application. The present study manifest novel insight about the alleviation of OTA-induced nephrotoxicity by nutrition regulation, and had a guiding effect on the clinical supplementation of nutritional elements.

Fumonisin B1 induces nephrotoxicity via autophagy mediated by mTORC1 instead of mTORC2 in human renal tubule epithelial cells.

Fumonisin B1 (FB1), a worldwide contaminating mycotoxin, can cause global food issue. It has been reported that FB1 is related to chronic kidney disease of unknown etiology. However, the study of FB1-induced nephrotoxicity in vitro is very limited and the mechanism is unknown. Human renal tubule epithelial (HK-2) cells were used in this study. The results showed that FB1 exposure could decrease cell viability, induce cell apoptosis and up-regulate the expression of Kim-1, collagen I, α-SMA and TGF-ß1. In addition, autophagy was activated after FB1 exposure, including the conversion of LC3 and up-regulation of ATGs. Furthermore, autophagy inhibitor 3-MA could block FB1-induced abnormalities. And antioxidant enzymes (Gpx1 and Gpx4) were obviously down-regulated and intracellular ROS levels displayed an ascent trend as FB1 exposure concentrations increased. Employing of antioxidant NAC could suppress FB1-induced nephrotoxicity and autophagy. FB1 inhibited the phosphorylation of p70 S6k, a downstream protein of mTORC1. Also, oxidative stress, autophagy and phosphorylation of p70 S6k induced by FB1 was inhibited by MHY1485, an activator of mTOR. But the phosphorylation of AKT, a downstream protein of mTORC2 showed no change with or without MHY1485. Taken together, FB1 induced nephrotoxicity via autophagy mediated by mTORC1 instead of mTORC2 in HK-2 cells.

Nontoxic dose of Phenethyl isothiocyanate ameliorates deoxynivalenol-induced cytotoxicity and inflammation in IPEC-J2 cells.

The intestinal tract is a target for the deoxynivalenol (DON), which has adverse effects in animals and humans' health by affecting intestinal functions. Phenethyl isothiocyanate (PEITC) is an important degradation product of glucosinolates (GSLs), belonging to an anti-nutritional factor that affects the digestion and absorption of nutrients in the animals' intestinal. However, little attention has been paid to the interaction and its mechanism between DON and PEITC. Therefore, the purpose of this study was to assess the effects of PEITC on DON-induced cytotoxicity and inflammation, and explore the potential mechanisms in IPEC-J2 cells. Our results showed that DON exposure could decrease the cell viability and pro-inflammatory cytokine expression in IPEC-J2 cells in a dose-dependent manner. PEITC treatment at the concentrations of 1.25-5 µM had no significant effect on IPEC-J2 cells viability, but above 10 µM of PEITC treatment significantly reduced the cell viability. Interestingly, 1.25-5 µM of PEITC treatment could suppress 4 µM of DON-induced decrease in cell viability and increase in pro-inflammatory cytokine expression. Meanwhile, the protein ratios of p-p65/p-65 and p-IκBα/IκBα were markedly decreased in the groups treated with 1.25-5 µM PEITC compared to DON exposure alone. However, the protective effects of PEITC treatment were significantly blocked after pre-treatment with LPS, NF-κB activator, in IPEC-J2 cells. In conclusion, these findings indicated that the nontoxic dose of PEITC could alleviate DON-induced cytotoxicity and inflammation responses via suppressing the NF-κB signaling pathway in IPEC-J2 cells. Our results provide a new theoretical basis for the rational addition of rapeseed meal in animal feedstuff.

Nontoxic-dose deoxynivalenol aggravates lipopolysaccharides-induced inflammation and tight junction disorder in IPEC-J2 cells through activation of NF-κB and LC3B.

Lipopolysaccharide (LPS) is the key factor in various intestinal inflammation which could disrupt the epithelial barrier function. Deoxynivalenol (DON), a well-known mycotoxin, can induce intestinal injury. However, the combined enterotoxicity of LPS and DON has rarely been studied. In this study, IPEC-J2 cell monolayers were exposed to LPS and nontoxic-dose DON for 12 and 24 h to investigate the effects of DON on LPS-induced inflammatory response and tight junction variation, and specific inhibitor and CRISPR-Cas9 were used to explore the underlying mechanisms. Our results showed that nontoxic-dose DON aggravated LPS-induced cellular inflammatory response, reflecting on more significant changes of inflammatory cytokines mRNA expression, higher protein expression of NOD-like receptor protein 3 (NLRP3) and procaspase-1. Moreover, nontoxic-dose DON aggravated LPS-induced mRNA and protein expression decreased, and distribution confused of tight junction proteins. We found that DON further enhanced LPS-induced phosphorylation and nucleus translocation of p65, and expression of LC3B-Ⅱ. NF-κB inhibitor and CRISPR-Cas9-mediated knockout of LC3B attenuated the effects of combination which indicated nontoxic-dose DON aggravated LPS-induced intestinal inflammation and tight junction disorder through activating NF-κB signaling pathway and autophagy-related protein LC3B. It further warns that ingesting low doses of mycotoxins may exacerbate the effects of intestinal pathogens on the body.

Ochratoxin A induces glomerular injury through activating the ERK/NF-κB signaling pathway.

Ochratoxin A (OTA) was reported to induce proximal tubules nephrotoxicity in humans and animals. However, the toxicity of OTA on glomeruli has rarely been studied. We investigated OTA-induced glomerular injury and the underlying mechanisms. Mice were intraperitoneally treated with OTA (0, 0.5, 1.5 and 2.5 mg/kg b.w.) on alternate day for 3 weeks. OTA exposure decreased the weight gain ratio, the kidney index and increased the levels of serum creatinine and blood urea nitrogen. It induced also fragmentation and atrophy in glomeruli, and increased the expression of TNF-α, IL-6, COX-2, TGF-ß, α-SMA and vimentin in a dose-dependent manner. Human mesangial cells (HMC) were treated with OTA (0-8 µM) for 48 h. Treatment of HMC cells with OTA increased cell inhibition rate, up-regulated the expression of IL-6, TGF-ß, α-SMA and vimentin in a dose-dependent manner. Additionally, it enhanced the phosphorylation of ERK1/2 and p65, degradation of IκB-α and translocation of p65 into the nucleus. OTA-induced toxicity was attenuated by NF-κB and ERK1/2 inhibitors. In conclusion, these results suggest that OTA exposure induces glomerular injury via activation of the ERK/NF-κB signaling pathway, and provide novel insights into the research of OTA induced nephrotoxicity.

Regulation of taurine in OTA-induced apoptosis and autophagy.

Ochratoxin A (OTA), one of the most deleterious mycotoxins, could cause a variety of toxicological effects especially nephrotoxicity in animals and humans. Taurine, a wide-distributed cytoprotective amino acid, plays an important role as a basic factor for maintaining cellular integrity homeostasis. However, the potential effect of taurine in OTA-induced nephrotoxicity remains unknown. In the present study, we demonstrated that OTA treatment at 4.0-8.0 µM increased apoptosis in PK-15 cells as shown by increased the ratio of apoptosis and protein expression of Bax and cleaved-caspase-3, decreased protein expression of Bcl-2. Meantime, OTA treatment triggered autophagy, as indicated by markedly increased the protein expression of LC3-II and fluorescence intensity of GFP-LC3 dots. Taurine supplementation decreased OTA-induced cytotoxicity and attenuated apoptosis as shown by the decreased Annexin V/PI staining and the decreased expression of apoptosis-related proteins including Bax and caspase-3. Meanwhile, taurine attenuated OTA-induced autophagy by decreased the protein expression of LC3-II and fluorescence intensity of GFP-LC3 dots to maintain cellular homeostasis. In conclusion, taurine treatment could alleviate OTA-induced apoptosis and inhibit the triggered autophagy in PK-15 cells. Our study provides supportive data for the potential roles of taurine in reducing OTA-induced renal toxicity.

Nontoxic concentration of ochratoxin A decreases the dosage of cyclosporine A to induce chronic nephropathy model via autophagy mediated by toll-like receptor 4.

Cyclosporine A (CsA) extracted from the products of fungal fermentation is used to develop a chronic nephropathy model. However, it has numerous side effects. Ochratoxin A (OTA) is a mycotoxin that induces renal injury. We developed a chronic nephropathy model to lessen the side effects of CsA by administration of nontoxic dosage of OTA, and investigated the underlying mechanism. C57BL/10 wild-type mice, toll-like receptor 4 (TLR4)-/- mice, and HK-2 cells were used in this study. The nontoxic dosage (0.25 mg/kg, qod) of OTA could significantly decrease the dosage of CsA from 30 to 20 mg/kg per day, and combination of them induced chronic nephropathy model and alleviated the side effects of onefold CsA in vivo, including cardiotoxicity, hepatotoxicity, and immunosuppression. The nontoxic concentration (0.5 µg/ml) of OTA could significantly decrease the concentration of CsA from 10 to 6 µg/ml that induced cytotoxicity, oxidative stress, and nephrotoxicity in vitro. Nontoxic concentration of OTA and low dosage of CsA activated TLR4 and autophagy. These toxic effects induced by OTA and CsA could be reversed by knockdown of TLR4 and autophagy inhibitor 3-methyladenine in vitro. Furthermore, the renal injury and autophagy induced by OTA and CsA could be attenuated in TLR4-/- mice. It suggested that a chronic nephropathy model had been successfully developed by administration of nontoxic concentration of OTA and low dosage of CsA via TLR4-mediated autophagy. The side effects of current model were significantly lesser than those of the previous model induced by onefold CsA. It provided a new tool for exploring the pathogenesis and treatment of chronic kidney disease.

Aflatoxin B1 Induces Immunotoxicity through the DNA Methyltransferase-Mediated JAK2/STAT3 Pathway in 3D4/21 Cells.

As the most toxic mycotoxin of all of the fungal toxins, aflatoxin B1 (AFB1) has carcinogenesis, heptotoxicity, and immunotoxicity. DNA methylation plays a critical role in gene expression regulation of the pathological process. However, the relationship between DNA methylation and AFB1-induced immunotoxicity was not yet reported. Therefore, the objectives of this study were to verify AFB1-induced immunotoxicity and investigate the potential role of the DNA methyltransferase (DNMT) family in AFB1-induced immunotoxicity and the pathway mechanism in 3D4/21 cells. The results showed that AFB1 could induce cytotoxicity, apoptosis, pro-inflammatory cytokine expression, DNA damage, and oxidative stress and decrease phagocytotic capacity. Meanwhile, the levels of DNMT1 and DNMT3a were significantly increased in 0.04 and 0.08 µg/mL AFB1 compared to the control. Inhibition of DNMT1 and DNMT3a by 5-Aza-2dc could reverse changes of the above parameters. Further, the JAK2/STAT3 pathway was significantly activated in 0.04 µg/mL AFB1. Inhibition of p-JAK2 and p-STAT3 by AG490 could alleviate AFB1-induced immunotoxicity. Moreover, inhibition of DNMT1 and DNMT3a by 5-Aza-2dc could suppress the phosphorylation of JAK2 and STAT3. Taken together, AFB1-induced immunotoxicity is related to the JAK2/STAT3 pathway mediated by DNMTs in 3D4/21 cells.