Ochratoxin A Treatment Research Articles

Mannan Oligosaccharide Could Attenuate Ochratoxin A-Induced Immunosuppression with Long-Time Exposure Instead of Immunostimulation with Short-Time Exposure

Our previous study showed that ochratoxin A (OTA), one of the most common mycotoxins in feed, could induce immunosuppression with long-time exposure but immunostimulation with short-time exposure. However, limited studies for the control of OTA-induced two-way immune toxicity were carried out. This study explored the effects of mannan oligosaccharide (MOS), a glucomannoprotein complex with immunoregulatory capability derived from the yeast cell wall, on OTA-induced immune toxicity and its underlying mechanisms. Surprisingly, the results showed that MOS significantly attenuated immunosuppression induced by long-time OTA treatment but did not provide protection against immunostimulation induced by short-time OTA treatment on porcine alveolar macrophages (PAMs), as demonstrated by the expressions of inflammatory cytokines and the capability of migration and phagocytosis. Further, MOS increased the OTA-inhibited autophagy level and the JNK phosphorylation level on PAMs with long-time OTA treatment. In addition, the inhibition of autophagy by 3-MA or the inhibition of JNK phosphorylation by SP600125 could partly block the protective effects of MOS on OTA-induced immunosuppression. Importantly, the inhibition of JNK phosphorylation down-regulated the MOS-promoted autophagy level. In conclusion, MOS could attenuate OTA-induced immunosuppression with short-time exposure on PAMs through activating JNK-mediated autophagy but had no significant effects on OTA-induced immunostimulation with short-time exposure. Our study provides new insights into the application of MOS as an immunoregulator against mycotoxin-induced immune toxicity.

Curcumin Modulates Nitrosative Stress, Inflammation, and DNA Damage and Protects against Ochratoxin A-Induced Hepatotoxicity and Nephrotoxicity in Rats.

Ochratoxin A (OTA) is a fungal toxin of critical concern for food safety both for human health and several animal species, also representing a cancer threat to humans. Curcumin (CURC) is a natural polyphenol that has anti-apoptotic, anti-inflammatory, and antioxidant effects. The aim of this study was to investigate the cytoprotective effect of CURC against OTA-induced nephrotoxicity and hepatotoxicity through the study of the nitrosative stress, pro-inflammatory cytokines, and deoxyribonucleic acid (DNA) damage. Sprague Dawley rats were daily treated with CURC (100 mg/kg b.w.), OTA (0.5 mg/kg b.w), or CURC with OTA by oral gavage for 14 days. Our results demonstrated that OTA exposure was associated with significant increase of pro-inflammatory and DNA oxidative-damage biomarkers. Moreover, OTA induced the inducible nitric oxide synthase, (iNOS) resulting in increased nitric oxide (NO) levels both in kidney and liver. The co-treatment OTA + CURC counteracted the harmful effects of chronic OTA treatment by regulating inflammation, reducing NO levels and oxidative DNA damage in kidney and liver tissues. Histology revealed that OTA + CURC treatment determinates mainly an Iba1+ macrophagic infiltration with fewer CD3+ T-lymphocytes in the tissues. In conclusion, we evidenced that CURC exerted cytoprotective and antioxidant activities against OTA-induced toxicity in rats.

Oral subchronic exposure to the mycotoxin ochratoxin A induces key pathological features of Parkinson's disease in mice six months after the end of the treatment

Some epidemiological studies with different levels of evidence have pointed to a higher risk of Parkinson's disease (PD) after exposure to environmental toxicants. A practically unexplored potential etiological factor is a group of naturally-occurring fungal secondary metabolites called mycotoxins. The mycotoxin ochratoxin A (OTA) has been reported to be neurotoxic in mice. To further identify if OTA exposure could have a role in PD pathology, Balb/c mice were orally treated with OTA (0.21, 0.5 mg/kg bw) four weeks and left for six months under normal diet. Effects of OTA on the onset, progression of alpha-synuclein pathology and development of motor deficits were evaluated. Immunohistochemical and biochemical analyses showed that oral subchronic OTA treatment induced loss of striatal dopaminergic innervation and dopaminergic cell dysfunction responsible for motor impairments. Phosphorylated alpha-synuclein levels were increased in gut and brain. LAMP-2A protein was decreased in tissues showing alpha-synuclein pathology. Cell cultures exposed to OTA exhibited decreased LAMP-2A protein, impairment of chaperone-mediated autophagy and decreased alpha-synuclein turnover which was linked to miRNAs deregulation, all reminiscent of PD. These results support the hypothesis that oral exposure to low OTA doses in mice can lead to biochemical and pathological changes reported in PD.

Time Course of Renal Transcriptomics after Subchronic Exposure to Ochratoxin A in Fisher Rats.

The mycotoxin ochratoxin A (OTA) is a potent nephrocarcinogen, mainly in male rats. The aim of this study was to determine the time course of gene expression (GeneChip® Rat Gene 2.0 ST Array, Affymetrix) in kidney samples from male and female F344 rats, treated daily (p.o) with 0.50 mg/kg b.w. (body weight) of OTA for 7 or 21 days, and evaluate if there were differences between both sexes. After OTA treatment, there was an evolution of gene expression in the kidney over time, with more differentially expressed genes (DEG) at 21 days. The gene expression time course was different between sexes with respect to the number of DEG and the direction of expression (up or down): the female response was progressive and consistent over time, whereas males had a different early response with more DEG, most of them up-regulated. The statistically most significant DEG corresponded to metabolism enzymes (Akr1b7, Akr1c2, Adh6 down-regulated in females; Cyp2c11, Dhrs7, Cyp2d1, Cyp2d5 down-regulated in males) or transporters (Slc17a9 down-regulated in females; Slco1a1 (OATP-1) and Slc51b and Slc22a22 (OAT) down-regulated in males). Some of these genes had also a basal sex difference and were over-expressed in males or females with respect to the other sex.

Microphysiological system modeling of ochratoxin A-associated nephrotoxicity

Ochratoxin A (OTA) is one of the most abundant mycotoxin contaminants in food stuffs and possesses carcinogenic, nephrotoxic, teratogenic, and immunotoxic properties. Specifically, a major concern is severe nephrotoxicity, which is characterized by degeneration of epithelial cells of the proximal tubules and interstitial fibrosis. However, the mechanism of OTA toxicity, as well as the genetic risk factors contributing to its toxicity in humans has been elusive due to the lack of adequate models that fully recapitulate human kidney function in vitro. The present study attempts to evaluate dose-response relationships, identify the contribution of active transport proteins that govern the renal disposition of OTA, and determine the role of metabolism in the bioactivation and detoxification of OTA using a 3D human kidney proximal tubule microphysiological system (kidney MPS). We demonstrated that LC50 values of OTA in kidney MPS culture (0.375–1.21 μM) were in agreement with clinically relevant toxic concentrations of OTA in urine. Surprisingly, no enhancement of kidney injury biomarkers was evident in the effluent of the kidney MPS after OTA exposure despite significant toxicity observed by LIVE/DEAD staining. Instead, these biomarkers decreased in an OTA concentration-dependent manner. Furthermore, the effect of 1-aminobenzotriazole (ABT) and 6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX), pan-inhibitors of P450 and glutathione S-transferase (GST) enzymes, respectively, on OTA-induced toxicity in kidney MPS was examined. These studies revealed significant enhancement of OTA-induced toxicity by NBDHEX (3 μM) treatment, whereas ABT (1 mM) treatment decreased OTA-induced toxicity, suggesting roles for GSTs and P450 enzymes in the detoxification and bioactivation of OTA, respectively. Analysis of transcriptional changes using RNA-sequencing of kidney MPS treated with different concentrations of OTA revealed downregulation of several nuclear factor (erythroid derived-2)-like 2 (NRF2)-regulated genes by OTA treatment, including GSTs. The transcriptional repression of GSTs is likely playing a key role in OTA toxicity via attenuation of glutathione conjugation/detoxification. The sequential molecular events may explain the mechanism of toxicity associated with OTA. Additionally, OTA transport studies using kidney MPS in the presence and absence of probenecid (1 mM) suggested a role for organic anionic membrane transporter(s) in the kidney specific disposition of OTA. Our findings provide a clearer understanding of the mechanism of OTA-induced kidney injury, which may support changes in risk assessment, regulatory agency policies on allowable exposure levels, and determination of the role of genetic factors in populations at risk for OTA nephrotoxicity.

Ochratoxin A induces epithelial-to-mesenchymal transition and renal fibrosis through TGF-β/Smad2/3 and Wnt1/β-catenin signaling pathways in vitro and in vivo.

Ochratoxin A (OTA) is a toxin produced by fungi such as Aspergillus spp. and Penicillium spp. The key target organ of OTA toxicity is the kidney, and it is known that epithelial-to-mesenchymal transition (EMT) leading to fibrosis is enhanced after long-term exposure of the kidney to OTA. However, the mechanisms responsible for this onset are not precisely known. Therefore, the purpose of this study was to investigate the mechanism of OTA-induced EMT and fibrosis in human proximal tubule HK-2 cells and mouse kidneys. Cells were treated for 48h with various concentrations of OTA (50, 100, and 200nM) and mice underwent oral administration of various doses of OTA (200 and 1000μg/kg body weight) for 12weeks. Blood urea nitrogen and creatinine levels were increased in the serum of OTA-treated mice, and fibrosis was observed in kidney tissues. Furthermore, alpha-smooth muscle actin (α-SMA) and fibronectin levels were increased, and E-cadherin level was decreased by OTA in both HK-2 cells and kidney tissues. In addition, the expression levels of TGF-β, smad2/3, and β-catenin were increased after OTA treatment. α-SMA, E-cadherin, and fibronectin were shown to be regulated by the activation of transcription factors, smad2/3 and β-catenin. These results demonstrated that OTA induces EMT and renal fibrosis through Smad2/3 and β-catenin signaling pathways in vitro and in vivo.

Short-term effects of deoxynivalenol, T-2 toxin, fumonisin B1 or ochratoxin on lipid peroxidation and glutathione redox system and its regulatory genes in common carp (Cyprinus carpio L.) liver

The effects of a single oral dose of 1.82 mg kg−1 bw of T-2 and HT-2 toxin (T-2), 1.75 mg kg−1 bw deoxynivalenol (DON) and 15-acetyl DON, 1.96 mg kg−1 bw fumonisin B1 (FB1) or 1.85 mg kg−1 bw ochratoxin A (OTA) were investigated in common carp juveniles on lipid peroxidation, the parameters of the glutathione redox system including the expression of their encoding genes in a short-term (24 h) experiment. Markers of the initiation phase of lipid peroxidation, conjugated dienes, and trienes, were slightly affected by DON and OTA treatment at 16-h sampling. The termination marker, malondialdehyde, concentration increased only as an effect of FB1. Glutathione content and glutathione peroxidase activity showed significantly higher levels in the T-2 and FB1 groups at 8 h, and in the DON and FB1 groups at 16 h. The expression of glutathione peroxidase genes (gpx4a, gpx4b) showed a dual response. Downregulation of gpxa was observed at 8 h, as the effect of DON, FB1, and OTA, but an upregulation in the T-2 group. At 16 h gpx4a upregulated as an effect of DON, T-2, and FB1, and at 24 h in the DON and T-2 groups. Expression of gpx4b downregulated at 8 h, except in the T-2 group, and upregulation observed as an effect of T-2 at 24 h. The lack of an increase in the expression of nrf2, except as the effect of DON at 8 h, and a decrease in the keap1 expression suggests that the antioxidant defence system was activated at gene and protein levels through Keap1–Nrf2 independent pathways.

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.

Protective effect of curcumin on ochratoxin A–induced liver oxidative injury in duck is mediated by modulating lipid metabolism and the intestinal microbiota

Curcumin has antioxidant functions, regulates the intestinal microbial composition, and alleviates mycotoxin toxicity. The present study aimed to explore whether curcumin could alleviate ochratoxin A (OTA)-induced liver injury via the intestinal microbiota. A total of 720 mixed-sex 1-day-old White Pekin ducklings were randomly assigned into 4 groups: CON (control group, without OTA), OTA (fed a diet with 2 mg/kg OTA), CUR (ducks fed a diet with 400 mg/kg curcumin), and OTA + CUR (2 mg/kg OTA plus 400 mg/kg curcumin). Each treatment consisted of 6 replicates and 30 ducklings per replicate. Treatment lasted for 21 D. Results were analyzed by a two-tailed Student t test between 2 groups. Our results demonstrated that OTA treatment had the highest serum low-density lipoprotein (LDL) level among 4 groups. Compared with OTA group, OTA + CUR decreased serum LDL level (P < 0.05). OTA decreased liver catalase (CAT) activity in ducks (P < 0.05), while addition of curcumin in OTA group increased liver CAT activity (P < 0.05). 16S ribosomal RNA sequencing suggested that curcumin increased the richness indices (ACE index) and diversity indices (Simpson index) compared with OTA group (P < 0.05) and recovered the OTA-induced alterations in composition of the intestinal microbiota. Curcumin supplementation relieved the decreased abundance of butyric acid producing bacteria, including blautia, butyricicoccus, and butyricimonas, induced by OTA (P < 0.05). OTA also significantly influenced the metabolism of the intestinal microbiota, such as tryptophan metabolism and glyceropholipid metabolism. Curcumin could alleviate the upregulation of oxidative stress pathways induced by OTA. OTA treatment also increased SREBP-1c expression (P < 0.05). The curcumin group had the lowest expression of FAS and PPARG mRNA (P < 0.05) and the highest expression of NRF2 and HMOX1 mRNA. These results indicated that curcumin could alleviate OTA-induced oxidative injury and lipid metabolism disruption by modulating the cecum microbiota.

Ochratoxin A exposure causes meiotic failure and oocyte deterioration in mice

Ochratoxin A (OTA) is a mycotoxin produced by fungi and occurs naturally in various foodstuffs and some animal-derived products. This mycotoxin can cause deleterious effects on kidney, liver, central nervous, and immune system. However, potential mechanisms regarding how OTA disrupts the mammalian oocyte quality have not been clearly defined. In this study, we proved that OTA weakened oocyte quality by impairing oocyte meiotic maturation. We found that female mice treated with 1 mg/kg body weight OTA by intraperitoneal (IP) injection for 7 days displayed ovarian dysfunction and decreased offspring number. We also found that OTA treatment at 7.5 μM for 16 h decreased the rate of first polar body extrusion by disrupting spindle and chromosome alignment. In addition, OTA caused oxidative stress by inducing the accumulation of reactive oxygen species and consumption of antioxidants during meiosis, consequently resulting in oocytes apoptosis. Mitochondrial damage and insufficient energy supply were also observed in OTA-pretreated oocytes, which led to the meiotic failure of oocyte. Moreover, the epigenetic modifications were also affected, showing with altered 5 mC, 5hmC, H3K9ac, and H3K9me3 levels in mice oocytes. In summary, these results showed that OTA could decrease oocyte maturation and fertility by inducing oxidative stress and epigenetic changes.

Amelioration of ochratoxin-A induced cytotoxicity by prophylactic treatment of N-Acetyl-L-Tryptophan in human embryonic kidney cells

Ochratoxin A (OTA) is known to induce nephro-toxicity via induction of cellular redox homeostasis perturbation, mitochondrial hyperpolarisation and depolarization, protein synthesis inhibition leading to apoptosis. In the present study, protective efficacy of N-Acetyl-L-Tryptophan (NAT) against OTA induced toxicity was evaluated using Human Embryonic Kidney (HEK-293) cells. Cells were treated with NAT (0–200 μg/ml) before OTA treatment (0–20 μg/ml) and protective efficacy of NAT was evaluated using MTT and SRB assay. OTA-induced intracellular ROS generation and its inhibition by NAT (2.5 μg/ml) pre-treatment was evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) probe. Effects of NAT pre-treatment on OTA treated cells were also evaluated in terms of cell cycle perturbations and mitochondrial membrane potential disturbance using flowcytometry. Results of the study demonstrated significant (∼89 % cell growth in comparison to 50% in OTA alone group; P < 0.05) protection by NAT to the HEK-293 cells against OTA mediated cell death in terms of cell viability. Further, significant reduction in ROS levels and mitochondrial membrane potential disturbance was also observed in NAT pre-treated plus OTA cells as compared to only OTA treated cells. Significant (p < 0.05) arrest in G0 and S phase of cell cycle was observed in OTA treated cells that was found to be inhibited by NAT pre-treatment to OTA treated cells. Also, molecular docking analysis demonstrated higher probability of NAT to bind with OTA binding pocket on phenylalanyl t-RNA synthetase, resulting in inhibition of OTA incorporation in the newly synthesized peptides and thus may ameliorate OTA induced protein synthesis inhibition. Conclusively, present study suggested that NAT offers protection against OTA toxicity in HEK-293 cells by counterbalancing oxidative stress, cell cycle regulation, mitochondrial membrane potential stabilization, protein synthesis inhibition and cell death retardation.

Melatonin ameliorates ochratoxin A-induced oxidative stress and apoptosis in porcine oocytes

Melatonin is a hormone which is generated from pineal gland, and it is responsible for the regulation of wake-sleep cycle. Melatonin is a well-known antioxidant and free radical scavenger to protect against multiple type of tissue damage. While ochratoxin A (OTA) is a mycotoxin found widely in contaminated food and foodstuffs, which causes nephrotoxicity, hepatotoxicity, immunotoxicity, and reproductive damage in humans and animals. In present study we report the toxicity of OTA on porcine oocyte quality and the protective effects of melatonin on OTA-exposed oocytes. Using transcriptome analysis our results show that OTA exposure alters the expression of multiple genes in oocytes, indicating its effect on oocyte maturation. The cellular changes following OTA treatment are examined, and the results show that OTA adversely affects oocyte polar body extrusion, which is confirmed by the delay of Cdc2-mediated cell cycle progression. OTA exposure also disrupts meiotic spindle formation, which is confirmed by altered phosphorylated MAPK expression. RNA-seq screening and further fluorescence staining results show that OTA induces aberrant mitochondria distribution and oxidative phosphorylation defects, which then causes oxidative stress, followed by early apoptosis and autophagy. Treatment with melatonin significantly ameliorates oxidative stress and apoptosis, which further protects cell cycle and spindle formation in OTA-exposed oocytes. Collectively, these results show the protective effects of melatonin against defects induced by OTA during porcine meiotic oocyte maturation.

Ochratoxin A induces global DNA hypomethylation and oxidative stress in neuronal cells in vitro.

Recently, it was reported that ochratoxin A (OTA) mycotoxin, produced by a number of Aspergillus and Penicillium fungal species, may cause neuropsychological impairment or mental and emotional disorders but the mechanism of neurotoxicity remains unknown. Adverse effects of OTA in human (SHSY5Y) and mouse (HT22) neuronal cell lines were studied in vitro. OTA was found to be non-cytotoxic in both cell lines at concentrations 2.5-30μmol/l, which are above the levels reported for human and animal plasma. OTA led to slightly elevated chromosomal instability in HT22 cells at concentrations of 15-30μmol/l after 48h, while in SHSY5Y cells, no evidence for genotoxic effects was observed at concentrations of 2.5-30μmol/l. OTA treatment at 10μmol/l resulted in elevated levels of unmethylated cytosines in CpG dinucleotides (up to 1.4-fold), elevated levels of intracellular reactive oxygen species (up to 1.6-fold), and in elevated levels of oxidized DNA purines (up to 2.2-fold) in both cell lines. Detected global DNA hypomethylation and oxidative stress were found to be reversible in 96h and 24-72h, respectively. In general, the observed pattern of OTA-induced effects in both cell lines was similar, but HT22 cells exhibited higher sensitivity, as well as better repair capacity in response to OTA toxicity. In conclusion, the results suggest that oxidative stress and epigenetic changes are directly involved in OTA-induced neurotoxicity, while cytotoxicity and genotoxicity cannot be considered as primary cause of toxicity in neuronal cells in vitro.

Ameliorative effect of ursolic acid on ochratoxin A-induced renal cytotoxicity mediated by Lonp1/Aco2/Hsp75

Ochratoxin A (OTA) is a mycotoxin ubiquitous in feeds and foodstuffs. The water-insoluble pentacyclic triterpene bioactive compound, ursolic acid (UA), is widespread in various cuticular waxes of edible fruits, food materials, and medicinal plants. Although studies have reported that oxidative stress was involved in both the nephrotoxicity of OTA and the renoprotective function of UA, the role of stress-responsive Lon protease 1 (Lonp1) in the renoprotection of UA against OTA is still unknown. In this study, cell viability, reactive oxygen species (ROS) production, and several proteins’ expressions of human embryonic kidney 293T (HEK293T) cells in response to UA, OTA, and/or Lonp1 inhibitor CDDO-me treatment were detected to reveal the protective mechanism of UA against OTA-induced renal cytotoxicity. Results indicated that a 2 h-treatment of 1 μM UA could significantly alleviate the ROS production and cell death induced by a 24 h-treatment of 8 μM OTA in HEK293T cells (P < 0.05). Compared with the control, the protein expressions of Lonp1, Aco2 and Hsp75 were significantly inhibited after 8 μM OTA treating for 24 h (P < 0.05), which could be notably reversed by the pre-treatment and post-treatment of 1 μM UA (P < 0.05). The protein expressions of Lonp1, Aco2 and Hsp75 were inhibited by the addition of CDDO-me. The three protein expression trends were similar before and after the addition of CDDO-me. In conclusion, OTA could inhibit the expression of Lonp1, suppressing Aco2 and Hsp75 as a result, thereby activating ROS and inducing cell death in HEK293T cells, which could be alleviated by UA pre-treatment.

Ochratoxin A-Induced Hepatotoxicity through Phase I and Phase II Reactions Regulated by AhR in Liver Cells.

Ochratoxin A (OTA) is a widespread mycotoxin produced by several species of the genera Aspergillus and Penicillium. OTA exists in a variety of foods, including rice, oats, and coffee and is hepatotoxic, with a similar mode of action as aflatoxin B1. The precise mechanism of cytotoxicity is not yet known, but oxidative damage is suspected to contribute to its cytotoxic effects. In this study, human hepatocyte HepG2 cells were treated with various concentrations of OTA (5–500 nM) for 48 h. OTA triggered oxidative stress as demonstrated by glutathione depletion and increased reactive oxygen species, malondialdehyde level, and nitric oxide production. Apoptosis was observed with 500 nM OTA treatment. OTA increased both the mRNA and protein expression of phase I and II enzymes. The same results were observed in an in vivo study using ICR mice. Furthermore, the relationship between phase I and II enzymes was demonstrated by the knockdown of the aryl hydrocarbon receptor (AhR) and NF-E2-related factor 2 (Nrf2) with siRNA. Taken together, our results show that OTA induces oxidative stress through the phase I reaction regulated by AhR and induces apoptosis, and that the phase II reaction is activated by Nrf2 in the presence of oxidative stress.

Cytotoxicity, Genotoxicity and Disturbance of Cell Cycle in HepG2 Cells Exposed to OTA and BEA: Single and Combined Actions.

Mycotoxins are produced by a number of fungal genera spp., for example, Aspergillus, Penicillium, Alternaria, Fusarium, and Claviceps. Beauvericin (BEA) and Ochratoxin A (OTA) are present in various cereal crops and processed grains. This goal of this study was to determine their combination effect in HepG2 cells, presented for the first time. In this study, the type of interaction among BEA and OTA through an isobologram method, cell cycle disturbance by flow cytometry, and genotoxic potential by in vitro micronucleus (MN) assay following the TG 487 (OECD, 2016) of BEA and OTA individually and combined in HepG2 cells are presented. Cytotoxic concentration ranges studied by the MTT assay over 24, 48, and 72 h were from 0 to 25 µM for BEA and from 0 to 100 µM for OTA, while BEA + OTA combinations were at a 1:10 ratio from 3.4 to 27.5 µM. The toxicity observed for BEA was higher than for OTA at all times assayed; additive and synergistic effects were detected for their mixtures. Cell cycle arrest in the G0/G1 phase was detected for OTA and BEA + OTA treatments in HepG2 cells. Genotoxicity revealed significant effects for BEA, OTA, and in combinations underlining the importance of studying real exposure scenarios of chronic exposure to mycotoxins.

Ochratoxin A causes mitochondrial dysfunction, apoptotic and autophagic cell death and also induces mitochondrial biogenesis in human gastric epithelium cells.

Ochratoxin A (OTA) is a common natural contaminant found in human and animal food worldwide. Our previous work has shown that OTA can cause oxidative DNA damage, G2 arrest and malignant transformation of human gastric epithelium (GES-1) cells. Mitochondria are considered to be target for the action of many cytotoxic agents. However, the role of mitochondria in the cytotoxicity of OTA remains unknown. The aim of this study is to explore the putative role of mitochondria on OTA cytotoxicity by analyzing mitochondrial changes in GES-1 cells. The results showed that OTA treatment (5, 10, 20µM) for different times caused increases in the production of reactive oxygen species, and induced mitochondrial damage, shown by loss of mitochondrial membrane potential (ΔΨM), and decrease in cellular ATP concentration. Subsequently, the mitochondrial apoptotic pathway was activated, presented by increase of apoptotic rate and activation of apoptotic proteins. Autophagic cell death was also triggered, demonstrated by the conversion of light chain 3B (LC3B)-I to LC3B-II and elevated levels of green fluorescent protein-LC3 (GFP-LC3) puncta. Moreover, Parkin-dependent mitophagy was also activated presented by the colocalization of MitoTracker with LysoTracker or GFP-LC3 puncta. The inhibition of autophagy and mitophagy by inhibitors or siRNA attenuated the toxic effect of OTA on cell growth. Interestingly, OTA treatment also enhanced mitochondrial biogenesis confirmed by activation of AMPK/PGC-1α/TFAM pathway and promoted cell survival. Collectively, the effects of OTA on mitochondria of GES-1 cells are complex. OTA could cause mitochondrial function disturbance, apoptotic and autophagic cell death and also induce mitochondrial biogenesis.

Inhibitory effect of ochratoxin A on DNMT-mediated flocculation of yeast.

The mycotoxin ochratoxin A (OTA) is considered to be a human carcinogen. However, the mode of its carcinogenetic action has not been elucidated. Recently, it has become evident that epigenetic changes influence the risk of developing cancer. Since it has been revealed that the yeast flocculation displayed by the strains transformed with human DNA methyltransferases (DNMT) can be regulated by epigenetic mechanisms, we examined the effect of OTA on the transcription level of FLO1, which mediates the flocculation phenotype. OTA but not a non-carcinogenetic mycotoxin deoxynivalenol (DON) inhibited the intensity of GFP fluorescence under the transcriptional regulation of FLO1 promoter in a dose-dependent manner. At the same time, OTA had no effect on the reporter activity under the control of modified FLO1 promoter with reduced CpG motifs. In addition, it was confirmed that the flocculation and FLO1 mRNA of DNMT gene-transformed yeast (DNMT yeast) were decreased by OTA. In vitro methylation assay using a bacterial DNMT revealed an inhibitory effect of OTA on the DNMT activity, and OTA treatment reduced the frequency of abnormally shaped nuclei which were often observed in DNMT yeast. These results suggest that the carcinogenicity of OTA may involve inhibition of DNMT-mediated epigenetic regulation.

Ochratoxin A Exposure Impairs Porcine Granulosa Cell Growth via the PI3K/AKT Signaling Pathway.

The mycotoxin ochratoxin A (OTA), a naturally occurring food contaminant, has a toxic effect on the growth and development of follicles in pigs. However, little is known regarding the specific toxic effects of OTA exposure on oocytes and granulosa cells (GCs). In this study, we cultured porcine ovarian GCs and exposed them to OTA in vitro in order to explore the mechanism causing the negative effects. Initially, it was found that OTA exposure inhibited cell viability in a time and dose dependent manner. We also showed that OTA exposure increased oxidative stress, decreased proliferation ratio, and increased apoptosis ratio in GCs. We revealed an important role for the PI3K/AKT signal pathway in GC proliferation and apoptosis by RNA-seq analysis. The results not only showed that OTA treatment significantly affected the expression of genes within the PI3K/AKT pathway but also demonstrated a concrete relationship between the PI3K/AKT pathway and GC cell proliferation and apoptosis. In conclusion, the results demonstrated that OTA exposure impaired porcine GC growth via the PI3K/AKT signaling pathway.

Oxidative Stress of in-Ovo Ochratoxin A Administered during Chick Embryonic Development

ABSTRACT The present work was carried out to study the effect of in-ovo injection of ochratoxin A (OTA) as an oxidative stress and its consequences on hepatic and kidney functions, thyroid activity, and histological examination of brain and liver in chicken embryos and subsequently in the hatching chicks. On the 10th day of incubation, one hundred and sixty-two fertile eggs were randomly divided into two equal treatments. Control treatment, (injected by 50 µl sodium carbonate) and OTA treatment (injected by 12.5 ng OTA dissolved in 50 µl sodium carbonate). OTA treatement group significantly reduced glutathione (GSH) and significantly increased thiobarbituric acid reactive substances production (TBARS) in embryonic and hatched chicks regarding livers, spleen, bursa of Fabricius, heart, and brain as an indicator of oxidative stress. OTA injection increased TBARS and decreased GSH levels in both allantoic and amniotic fluids. On the 14th and 16th days of incubation and at the hatch, a significant lower concentration in cholesterol and higher concentrations of alanine amino transferase, aspartate amino transferase, alkaline phosphatase, gamma glutamyl transferase, acid phosphatase enzymes activities and triglycerides in the hepatic tissues of the OTA group were observed. Histological examination of OTA group of brain and liver tissues showed some degenerative changes through the experimental period. In conclusion, in-ovo OTA treated had teratogenic and embryotoxic effects.