Identification of Biotransformation Products of T-2 Toxin in HepG2 Cells Using LC-Q-TOF MS.

The T-2 toxin (T-2) is a type A trichothecene found in cereals. The formation of metabolites is a frequent cause of mycotoxin-induced toxicity. In this work, the conversion of T-2 during biotransformation reactions in HepG2 cells was evaluated. For this, HepG2 cells were exposed to 30 (IC50/2) and 60 (IC50) nM of T-2 for 0, 1, 2, 3, 6, 8 and 24 h, and the concentrations of T-2 and its metabolites HT-2, T2-triol, T2-tetraol and neosolaniol were determined in both the cell fraction and culture medium through liquid chromatography coupled to high-resolution mass spectrometry-time of flight (LC-Q-TOF MS). Results showed a fast metabolization of T-2 (>90%) during the first 2 h, with HT-2 as its main (>95%) biotransformation product. The cell fraction showed higher levels (p < 0.05) of HT-2 (39.9 ± 2.1 nM) compared to the culture medium (12.53 ± 2.4 nM). This trend was also observed for the identified metabolites. T2-triol reached its maximum concentration (1.7 ± 0.4 nM) at 2 h, and at later times a time-dependent increase in the T2-tetraol and neosolaniol concentrations was observed. The identification of T-2 metabolites shows the need to continue combined toxicity studies of mycotoxins for a correct risk characterization of these natural contaminants.

Comparative Study of Spheroids (3D) and Monolayer Cultures (2D) for the In Vitro Assessment of Cytotoxicity Induced by the Mycotoxins Sterigmatocystin, Ochratoxin A and Patulin.

Mycotoxins are secondary metabolites produced by filamentous fungi associated with a variety of acute and chronic foodborne diseases. Current toxicology studies mainly rely on monolayer cell cultures and animal models, which are undeniably affected by several limitations. To bridge the gap between the current in vitro toxicology approach and the in vivo predictability of the data, we here investigated the cytotoxic effects induced by the mycotoxins sterigmatocystin (STE), ochratoxin A (OTA) and patulin (PAT) on different 2D and 3D cell cultures. We focused on human tumours (neuroblastoma SH-SY5Y cells and epithelial breast cancer MDA-MB-213 cells) and healthy cells (bone marrow-derived mesenchymal stem cells, BM-MSC, and umbilical vein endothelial cells, HUVECs). The cytotoxicity of STE, OTA, and PAT was determined after 24, 48 and 72 h of exposure using an ATP assay in both culture models. Three-dimensional spheroids' morphology was also analysed using the MATLAB-based open source software AnaSP 1.4 version. Our results highlight how each cell line and different culture models showed specific sensitivities, reinforcing the importance of using more complex models for toxicology studies and a multiple cell line approach for an improved and more comprehensive risk assessment.

Harmaline to Human Mitochondrial Caseinolytic Serine Protease Activation for Pediatric Diffuse Intrinsic Pontine Glioma Treatment.

Diffuse intrinsic pontine glioma (DIPG), affecting children aged 4-7 years, is a rare, aggressive tumor that originates in the pons and then spreads to nearby tissue. DIPG is the leading cause of death for pediatric brain tumors due to its infiltrative nature and inoperability. Radiotherapy has only a palliative effect on stabilizing symptoms. In silico and preclinical studies identified ONC201 as a cytotoxic agent against some human cancer cell lines, including DIPG ones. A single-crystal X-ray analysis of the complex of the human mitochondrial caseinolytic serine protease type C (hClpP) and ONC201 (PDB ID: 6DL7) allowed hClpP to be identified as its main target. The hyperactivation of hClpP causes damage to mitochondrial oxidative phosphorylation and cell death. In some DIPG patients receiving ONC201, an acquired resistance was observed. In this context, a wide program was initiated to discover original scaffolds for new hClpP activators to treat ONC201-non-responding patients. Harmaline, a small molecule belonging to the chemical class of ß-carboline, was identified through Fingerprints for Ligands and Proteins (FLAP), a structure-based virtual screening approach. Molecular dynamics simulations and a deep in vitro investigation showed interesting information on the interaction and activation of hClpP by harmaline.

The Growing Importance of Three-Dimensional Models and Microphysiological Systems in the Assessment of Mycotoxin Toxicity.

Current investigations in the field of toxicology mostly rely on 2D cell cultures and animal models. Although well-accepted, the traditional 2D cell-culture approach has evident drawbacks and is distant from the in vivo microenvironment. To overcome these limitations, increasing efforts have been made in the development of alternative models that can better recapitulate the in vivo architecture of tissues and organs. Even though the use of 3D cultures is gaining popularity, there are still open questions on their robustness and standardization. In this review, we discuss the current spheroid culture and organ-on-a-chip techniques as well as the main conceptual and technical considerations for the correct establishment of such models. For each system, the toxicological functional assays are then discussed, highlighting their major advantages, disadvantages, and limitations. Finally, a focus on the applications of 3D cell culture for mycotoxin toxicity assessments is provided. Given the known difficulties in defining the safety ranges of exposure for regulatory agency policies, we are confident that the application of alternative methods may greatly improve the overall risk assessment.

Climate Change and Effects on Molds and Mycotoxins.

Earth's climate is undergoing adverse global changes as an unequivocal result of anthropogenic activity. The occurring environmental changes are slowly shaping the balance between plant growth and related fungal diseases. Climate (temperature, available water, and light quality/quantity; as well as extreme drought, desertification, and fluctuations of humid/dry cycles) represents the most important agroecosystem factor influencing the life cycle stages of fungi and their ability to colonize crops, survive, and produce toxins. The ability of mycotoxigenic fungi to respond to Climate Change (CC) may induce a shift in their geographical distribution and in the pattern of mycotoxin occurrence. The present review examines the available evidence on the impact of CC factors on growth and mycotoxin production by the key mycotoxigenic fungi belonging to the genera Aspergillus, Penicillium, and Fusarium, which include several species producing mycotoxins of the greatest concern worldwide: aflatoxins (AFs), ochratoxins, and fumonisins (FUMs).

Development of an in vitro neuroblastoma 3D model and its application for sterigmatocystin-induced cytotoxicity testing.

Given the increasing importance of establishing better risk assessments for mycotoxins, novel in vitro tools for the evaluation of their toxicity are mandatory. In this study, an in vitro 3D spheroid model from SH-SY5Y cells, a human neuroblastoma cell line, was developed, optimized and characterized to test the cytotoxic effects caused by the mycotoxin sterigmatocystin (STE). STE induced a concentration- and time-dependent cell viability decrease in spheroids. Spheroids displayed cell disaggregation after STE exposure, increasing in a dose-dependent manner and over time. STE also induced apoptosis as confirmed by immunofluorescence staining and Western blot. Following the decreased proliferation and increased apoptosis, STE cytostasis effects were observed by migration assays both in 2D and 3D cell culture. Increased ROS generation, as well as DNA damage were also observed. Taken together, these data highlight the cytotoxic properties of STE and suggest that cell culture models play a pivotal role in the toxicological risk assessment of mycotoxins. The evaluation of cytotoxicity in spheroids (3D) rather than monolayer cultures (2D) is expected to more accurately reflect in vivo-like cell behaviour.

Role of quercetin on sterigmatocystin-induced oxidative stress-mediated toxicity.

Oxidative stress appears to be a common trigger for many of the effects associated with the exposure to various mycotoxins, including sterigmatocystin (STE). However, studies to alleviate STE toxicity through the use of natural antioxidants are sparsely reported in literature. In the present study, the cytoprotective effect of quercetin (QUE) was tested in SH-SY5Y cells against STE-induced oxidative stress and cytotoxicity. The MTT assay revealed that STE decreased cell viability, whereas pre-treatment of cells with QUE restored it. The QUE was also found to counteract STE-induced ROS generation and decrease STE-induced up-regulation of the expression of the stress-inducible enzymes HO-1 and NOS-2. Pre-treatment with QUE also prevented STE-induced nuclear translocation of NF-κB, as measured by immunofluorescence. Finally, considering the key role played by NF-κB in the regulation of inflammation, the effect of STE on the pro-inflammatory cytokines TNF-α and IL-6 expression was evaluated. Our results showed the down-regulation of TNF-α and IL-6 following STE exposure, suggesting a negative immunomodulatory effect of STE. In QUE pre-treated samples, TNF-α and IL-6 were significantly further reduced, indicating the anti-inflammatory role of QUE. The results of the present study demonstrate for the first time that QUE exerts a cytoprotective role in STE-induced toxicity.

Sterigmatocystin-induced DNA damage triggers cell-cycle arrest via MAPK in human neuroblastoma cells.

Sterigmatocystin (STE) is a common mycotoxin found in food and feed. Many studies showed that STE is genotoxic. However, up to now, the potential genotoxicity of STE on human neuronal system remains unknown. In this study, we explored the effect of STE on DNA damage and cell-cycle progression on human neuroblastoma SH-SY5Y cells exposed to various concentrations of STE (0.78, 1.56 and 3.12 µM) for 24 h. The results indicated that STE exposure induced DNA damage, as evidenced by DNA comet tails formation and increased γH2AX foci. Additionally, genotoxicity was confirmed by micronuclei (MN) analysis. Furthermore, we found that STE exposure led to cell-cycle arrest at the S and the G2/M phase. Considering the important role played by MAPK and p53 signaling pathways in cell-cycle arrest, we explored their potential involvement in STE-induced cell-cycle arrest by using specific inhibitors. The inhibition of JNK and ERK resulted to attenuate S and G2/M arrest, whereas the inhibition of p38 and p53 attenuated only STE-induced S phase arrest. In conclusion, the present study demonstrates that STE induced DNA damage and triggered MAPK and p53 pathways activation, resulting in cell-cycle arrest at the S and the G2/M phase.

Pharmacological and Genetic Evidence of Dopamine Receptor 3-Mediated Vasoconstriction in Isolated Mouse Aorta.

Dopamine receptors (DRs) are generally considered as mediators of vasomotor functions. However, when used in pharmacological studies, dopamine and/or DR agonists may not discriminate among different DR subtypes and may even stimulate alpha1 and beta-adrenoceptors. Here, we tested the hypothesis that D2R and/or D3R may specifically induce vasoconstriction in isolated mouse aorta. Aorta, isolated from wild-type (WT) and D3R-/- mice, was mounted in a wire myograph and challenged with cumulative concentrations of phenylephrine (PE), acetylcholine (ACh), and the D3R agonist 7-hydrxy-N,N-dipropyl-2-aminotetralin (7-OH-DPAT), with or without the D2R antagonist L741,626 and the D3R antagonist SB-277011-A. The vasoconstriction to PE and the vasodilatation to ACh were not different in WT and D3R-/-; in contrast, the contractile responses to 7-OH-DPAT were significantly weaker in D3R-/-, though not abolished. L741,626 did not change the contractile response induced by 7-OH-DPAT in WT or in D3R-/-, whereas SB-277011-A significantly reduced it in WT but did not in D3R-/-. D3R mRNA (assessed by qPCR) was about 5-fold more abundant than D2R mRNA in aorta from WT and undetectable in aorta from D3R-/-. Following transduction with lentivirus (72-h incubation) delivering synthetic microRNAs to specifically inactivate D2R (LV-miR-D2) or D3R (LV-miR-D3), the contractile response to 7-OH-DPAT was unaffected by LV-miR-D2, while it was significantly reduced by LV-miR-D3. These data indicate that, at least in mouse aorta, D3R stimulation induces vasoconstriction, while D2R stimulation does not. This is consistent with the higher expression level of D3R. The residual vasoconstriction elicited by high concentration D3R agonist in D3R-/- and/or in the presence of D3R antagonist is likely to be unrelated to DRs.

Occurrence, mitigation and in vitro cytotoxicity of nivalenol, a type B trichothecene mycotoxin - Updates from the last decade (2010-2020).

The present review aims to give an overview of the literature of the last decade (2010-2020) concerning the occurrence of the type B trichothecene mycotoxin nivalenol (NIV) and its in vitro toxicity, with the purpose of updating information regarding last researches on this mycotoxin. The most recent studies on the possible methods for preventing Fusarium spp. growth and NIV production are also discussed. Recently, various environmental factors have been shown to influence strongly NIV occurrence. However, Fusarium spp. of the NIV genotype have been found almost worldwide. With regard to NIV cytotoxicity, NIV has been reported to cause a marked decrease in cell proliferation in different mammalian cells. In particular, the recent data suggest that organs containing actively proliferating cells represent the main targets of NIV. Moreover, NIV resulted to cause immunosuppression, gastrointestinal toxicity and genotoxicity. However, sufficient evidence of carcinogenicity in humans is currently lacking, and the International Agency for Research on Cancer (IARC) classifies it as a group 3 carcinogen. Further researches and the discovery of effective treatment strategies to prevent NIV contamination and to counteract its toxicity are urgently required against this common food-borne threat to human health and livestock.

Sterigmatocystin: Occurrence, toxicity and molecular mechanisms of action - A review.

The mycotoxin sterigmatocystin (STE) is produced mainly by Aspergillus fungi. It has been reported to occur in grains and grain-based products, cheese, coffee, spices and beer. The STE is a known biogenic precursor of aflatoxin B1, sharing with it several structural and biological similarities. The STE has been shown to be hepatotoxic and nephrotoxic in animals and it has been classified as possible human carcinogen (group 2B) by IARC. The STE has been reported to cause a marked decrease in cell proliferation in different mammalian cells. Data available on literature suggest that the cellular mechanisms underlying STE-induced toxicity include the induction of oxidative stress, mitochondrial dysfunction, apoptosis, cell cycle arrest, as well as alteration of immune system function and activation of different signalling pathways. Moreover, STE resulted to be genotoxic, being able to form DNA-adducts and induce DNA damage. Despite its strong cytotoxicity, no risk assessments have been still carried out by authorities due to the lack of toxicity data, so research on STE toxicological impact is still going on. This review reports information available regarding STE toxicity and its related mechanisms of action with the aim of updating information regarding last researches on this mycotoxin.

The role of mitochondria in sterigmatocystin-induced apoptosis on SH-SY5Y cells.

Mitochondria are cellular organelles involved in many crucial functions, such as generation of energy (ATP) and initiation of apoptosis. The aim of the present study was to evaluate the role of mitochondria in the toxicity induced by sterigmatocystin (STE), a mycotoxin produced by fungi of the genus Aspergillus, on SH-SY5Y cells. Our results showed that STE exposure decreased cell viability in a time- and concentration-dependent manner by MTT assay and caused mitochondrial dysfunction, as highlighted by the increase of STE cytotoxicity in cells forced to rely on mitochondrial oxidative phosphorylation. Furthermore, intracellular ATP depletion and increased mitochondrial reactive oxygen species were also observed. Since mitochondria play a pivotal role in apoptosis, the induction of this process in response to STE exposure was decided to study. Our results showed an increase in apoptotic cell population by flow cytometry, further confirmed by the up-regulation of the expression levels of the pro-apoptotic genes Bax and Casp-3 and the down-regulation of the anti-apoptotic gene Bcl-2 by qPCR technique. Taken together, our results provide novel insights in the signalling pathways of the cell death process induced by STE in SH-SY5Y cells, highlighting the key role played by mitochondria in STE toxicity.

Cytotoxic effects of individual and combined sterigmatocystin and nivalenol on liver hepatocellular carcinoma cells.

Since humans are exposed to different mycotoxins through daily intake, there is increasing concern about the adverse effects of the interactions between them. Cytotoxicity of sterigmatocystin (STE) and nivalenol (NIV) alone and in combination in human hepatocarcinoma (HepG2) cells was evaluated by MTT assay. Furthermore, ROS production and alteration of ΔΨm as mechanisms of action were assessed. Cells were treated with concentrations ranging from 0.15 to 5 µM for NIV and from 0.78 to 50 µM for STE individually and in binary combinations. The combination ratio between the mixture STE + NIV was 10:1. The IC50 values of NIV ranged from 0.96 to 0.66 µM, whereas no IC50 values were obtained for STE at any time tested. For the combinations studied, synergistic, antagonistic and addictive effects were obtained with the two type of analyses performed, the isobologram analysis and the Combenefit method. No relevant effects on ROS and ΔΨm were observed. In conclusion, predictive models based on combination data could help to better understand the interactions between mycotoxins and their implications in food safety assessment. However, a further analysis of the molecular mechanism underlying these interactive effects is required.

Sterigmatocystin-induced cytotoxicity via oxidative stress induction in human neuroblastoma cells.

Sterigmatocystin (STE) is a mycotoxin produced by fungi of the genus Aspergillus. Considering that the effect of STE on neuronal system has not been well studied, the aim of the present study consists to investigate the cytotoxic effects of STE in human neuroblastoma (SH-SY5Y) cells. Moreover, the role of oxidative stress and intracellular defense systems was assessed by evaluating reactive oxygen species (ROS) generation, lipid peroxidation (LPO) and antioxidant no-enzymatic (GSH) levels and enzymatic (GPx, GST, CAT and SOD) activity. Our results revealed that STE decreased cell viability in a dose and time-dependent manner. Furthermore, after 24 h of exposure, STE induced an increase in ROS generation and LPO at all concentrations tested (0.78, 1.56 and 3.12 µM), as well as a depletion of GSH levels, an increase in GSSG content and a decrease in GSH/GSSG ratio at the highest concentrations. The activity of all antioxidant enzymes resulted to be also decreased. Additionally, an enhance of the oxidative damage was caused by BSO, a GSH depletor, while NAC, a GSH precursor, showed a scavenger activity. Our findings suggest that STE could injure SH-SY5Y cells via oxidative stress and highlight the antioxidant role of the glutathione system.

Heme Oxygenase-1 Inhibition Sensitizes Human Prostate Cancer Cells towards Glucose Deprivation and Metformin-Mediated Cell Death.

High levels of heme oxygenase (HO)-1 have been frequently reported in different human cancers, playing a major role in drug resistance and regulation of cancer cell redox homeostasis. Metformin (MET), a drug widely used for type 2 diabetes, has recently gained interest for treating several cancers. Recent studies indicated that the anti-proliferative effects of metformin in cancer cells are highly dependent on glucose concentration. The present work was directed to determine whether use of a specific inhibitor of HO-1 activity, alone or in combination with metformin, affected metastatic prostate cancer cell viability under different concentrations of glucose. MTT assay and the xCELLigence system were used to evaluate cell viability and cell proliferation in DU145 human prostate cancer cells. Cell apoptosis and reactive oxygen species were analyzed by flow cytometry. The activity of HO-1 was inhibited using a selective imidazole-based inhibitor; genes associated with antioxidant systems and cell death were evaluated by qRT-PCR. Our study demonstrates that metformin suppressed prostate cancer growth in vitro and increased oxidative stress. Disrupting the antioxidant HO-1 activity, especially under low glucose concentration, could be an attractive approach to potentiate metformin antineoplastic effects and could provide a biochemical basis for developing HO-1-targeting drugs against solid tumors.

Metformin: A Bridge between Diabetes and Prostate Cancer.

Prostate cancer (PCa) has become the most frequent type of cancer in men. Recent data suggest that diabetic patients taking metformin have a lower incidence of certain cancer, including PCa. Metformin is the most common drug used in type II diabetes mellitus; its use has been shown to lower the incidence of several cancers, although there are ambiguous data about the anticancer activity of metformin. A large number of studies examined the potential antineoplastic mechanism of metformin although it is not still completely understood. This review summarizes the literature concerning the effects of metformin on PCa cells, highlighting its numerous mechanisms of action through which it can act. We analyze the possible causes of the discordances regarding the impact of metformin on risk of PCa; we discuss the latest findings in this field, suggesting that metformin may have a future role in the management of PCa both as monotherapy and in combination with other drugs.