Daphnia magna model for the study of mycotoxins present in food: Gliotoxin, ochratoxin A and its combination.

Mycotoxins are low molecular weight compounds present in food and feed. Although their effects on human health have been widely described, their mechanisms of action are still undefined. Gliotoxin (GTX) and ochratoxin A (OTA) are among the most dangerous mycotoxins produced by Aspergillus spp. Therefore, their toxicity was studied in the Daphnia magna model, which has high capacity to predict cytotoxicity and assess ecotoxicity, comparable to mammalian models. The study consisted of a series of tests to evaluate the effects of mycotoxins GTX, OTA and their combinations at different dilutions on Daphnia magna that were conducted according to standardized OECD 202 and 211 guidelines. The following assays were carried out: acute toxicity test, heartbeat, delayed toxicity test, reproduction, growth rate test. Reproducibility was determined by observing the offspring after 21 days of GTX exposure. In acute and delayed toxicity transcript levels of genes involved in xenobiotic metabolism (mox, gst, abcb1, and abcc5), and oxidative stress (vtg-SOD) were analyzed by qPCR. GTX showed acute toxicity and decreased heart rate in D. magna compared to OTA. On the other hand, OTA showed a delayed effect as evidenced by the immobility test. Both mycotoxins showed to increase genes involved in xenobiotic metabolism, while only the mycotoxin mixture increased oxidative stress. These results suggest that the mycotoxins tested could have negative impact on the environment and human health.

Study of cytotoxicity in neuroblastoma cell line exposed to patulin and citrinin.

Mycotoxins can be found in food and feed storage as well as in several kinds of foodstuff and are capable of harming mammals and some of them even in small doses. This study investigated on the undifferentiated neuronal cell line SH-SY5Y the effects of two mycotoxins: patulin (PAT) and citrinin (CTN), which are predominantly produced by fungi species Penicillium and Aspergillus. Here, the individual and combined cytotoxicity of PAT and CTN was investigated using the cytotoxic assay MTT. Our findings indicate that after 24 h of treatment, the IC50 value for PAT is 2.01 µM, which decreases at 1.5 µM after 48 h. In contrast, CTN did not attain an IC50 value at the tested concentration. Therefore, we found PAT to be the more toxic compared to CTN. However, the combined treatment suggests an additive toxic effect. With 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) DCFH-DA assay, ROS generation was demonstrated after CTN treatment, but PAT showed only small changes. The mixture presented a very constant behavior over time. Finally, the median-effect/combination index (CI-) isobologram equation demonstrated an additive effect after 24 h, but an antagonistic effect after 48 h for the interaction of the two mycotoxins.

Involvement of pro-inflammatory mediators and cell cycle disruption in neuronal cells induced by gliotoxin and ochratoxin A after individual and combined exposure.

Mycotoxins such as gliotoxin (GTX) and ochratoxin A (OTA) are secondary metabolites of Aspergillus and Penicillum found in food and feed. Both mycotoxins have shown to exert a detrimental effect on neuronal activity. The following study was carried out to elucidate the mechanisms by which GTX and OTA exert their toxicity. Non-differentiated SH-SY5Y neuronal-like cells were treated with GTX, OTA and their combinations to assess their cytotoxic effect using the MTT assay during 24, 48 and 72 h of exposure. Based on the results of the cytotoxic assays, cell cycle proliferation and immunological mediators were measured by determining the production of IL-6 and TNF-α using flow cytometry and ELISA, respectively. The IC50 values obtained were 1.24 and 1.35 µM when SH-SY5Y cells were treated with GTX at 48 h and 72 h, respectively. IC50 values of 8.25, 5.49 and 4.5 µM were obtained for OTA treatment at 24 h, 48 h and 72 h, respectively. The SubG0 phase increased in both treatments at 24 and 48 h. On the other hand, IL-6 and TNF-α production was increased in all mycotoxin treatments studied and was more pronounced for [GTX + OTA] after 48 h exposure. The additive and synergistic effect observed by the isobologram analysis between GTX and OTA resulted to a higher cytotoxicity which can be explained by the increased production of IL-6 and TNF-α inflammatory mediators that play an important role in the toxicity mechanism of these mycotoxins.

Cell cycle and enzymatic activity alterations induced by ROS production in human neuroblastoma cells SH-SY5Y exposed to Fumonisin B1, Ochratoxin A and their combination.

The presence of mycotoxins such as Fumonisin B1(FB1) and Ochratoxin A (OTA) in food and feed has become a threat to human and animal health since they can produce several afflictions. Different mechanisms of action by which they exercise their cytotoxic activity have been attributed to them, including the production of reactive oxygen species (ROS). For this reason, a measurement of the production of ROS species, and an evaluation of the intrinsic cell enzymatic antioxidant activity, including glutathione peroxidase (GPx), glutathione transferase (GTS), and catalase (CAT) together with a cytotoxicity and cell cycle assay have been performed in undifferentiated SH-SY5Y cells exposed to FB1, OTA and [FB1 + OTA] after 24 h and 48 h. FB1 and OTA. Monitoring of intracellular ROS production was carried out by the H2-DCFDA probe; while spectrometry analysis of absorbances was used for measuring GPx, GST and CAT activity. Finally, cell proliferation and cell cycle distribution were studied by flow cytometry. When cells were treated with OTA, an increase in GPx and GST activity was observed compared to FB1 and [FB1 + OTA]; conversely, a decrease in CAT activity was observed when cells were exposed to OTA coinciding with the results observed for ROS measurement. Regarding the cell cycle, when cells were exposed to OTA, a decrease in G0/G1 was detected, revealing an arrest of cell division for SH-SY5Y cells at the concentrations studied.

Effectiveness of beetroot extract in SH-SY5Y neuronal cell protection against Fumonisin B1, Ochratoxin A and its combination.

Fumonisin B1 (FB1) and ochratoxin A (OTA) are fungal metabolites of worldwide concern because of their effect on human and animal health, as both have been classified by IARC as possible carcinogens (Group 2B). Beetroot is a source of dietary fiber, folic acid, and vitamin C, and some studies have demonstrated their antioxidant activity. Therefore, this work presents the cytoprotective effect of beetroot extract (BRE) on a neuroblastoma cell line (SH-SY5Y cells) exposed to FB1, OTA, and its combination. Cytotoxicity was studied by the MTT ([3-4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, for 24 h and 48 h. Simultaneous treatment and pre-treatment strategies were tested with 1:512-1:2 and 1:0 dilutions of BRE, with a concentration range from 0.4 to 100 µM of FB1 and from 0.19 to 50 µM of OTA. IC50 values of 5.8 µM and 9.1 µM at 24 h and 48 h, respectively were obtained for OTA while no cytotoxic effect was detected at the concentrations tested for FB1. Cytoprotection with increased viability was obtained when the simultaneous BRE + OTA strategy was performed. Finally, better protection was observed in the pretreatment strategy in which cells were exposed 24 h previously to BRE, compared to that shown in the simultaneous assay.

Effects of Voghiera garlic extracts in neuronal human cell line against zearalenone's derivates and beauvericin.

The Fusarium toxins constitute one of the largest groups of mycotoxins produced by Fusarium species, which are major pathogens of cereal plants. In the present study neuroprotection effect of Allium sativum L garlic extract which is known as Voghiera garlic, from a local garlic ecotype of Ferrara (Italy) was examined on an undifferentiated SH-SY5Y neuronal cells against ZEA's metabolites (α-zearalenol (α-ZEL) and ß-zearalenol (ß-ZEL)) and beauvericin (BEA) mycotoxins which are considered as the most reported Fusarium mycotoxins, via MTT (3-4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, over 24 h and 48 h through direct treatment, simultaneous treatment and pre-treatment strategies. The results demonstrated remarkable improvement in cells viability in simultaneous and pre-treatment strategy with Voghiera garlic extract (VGE); specifically, for simultaneous treatment of VGE with ß-ZEL which viability increased significantly up to 56%, and subsequently with α-ZEL and BEA by up to 38% and 37% respectively, compared to each mycotoxin tested alone for their highest concentrations assayed, while direct treatments for each mycotoxins individually decreased significantly (for α-ZEL up to 69%, for ß-ZEL 82% and for BEA up to 43%). It is proposed by the present study that VGE extract found to be effective in reducing the cytotoxicity/neurotoxicity of α-ZEL, ß-ZEL and BEA mycotoxins encountered in food and feed commodity.