Using Microfluidic Hepatic Spheroid Cultures to Assess Liver Toxicity of T-2 Mycotoxin.

The Fusarium fungi is found in cereals and feedstuffs and may produce mycotoxins, which are secondary metabolites, such as the T-2 toxin (T-2). In this work, we explored the hepatotoxicity of T-2 using microfluidic 3D hepatic cultures. The objectives were: (i) exploring the benefits of microfluidic 3D cultures compared to conventional 3D cultures available commercially (Aggrewell plates), (ii) establishing 3D co-cultures of hepatic cells (HepG2) and stellate cells (LX2) and assessing T-2 exposure in this model, (iii) characterizing the induction of metabolizing enzymes, and (iv) evaluating inflammatory markers upon T-2 exposure in microfluidic hepatic cultures. Our results demonstrated that, in comparison to commercial (large-volume) 3D cultures, spheroids formed faster and were more functional in microfluidic devices. The viability and hepatic function decreased with increasing T-2 concentrations in both monoculture and co-cultures. The RT-PCR analysis revealed that exposure to T-2 upregulates the expression of multiple Phase I and Phase II hepatic enzymes. In addition, several pro- and anti-inflammatory proteins were increased in co-cultures after exposure to T-2.

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.

Assessment of the genotoxic and mutagenic effects induced by T-2 mycotoxin in HepG2 cells.

The T-2 toxin is a mycotoxin produced by molds belonging to Fusarium. Among the Fusarium mycotoxins, trichothecenes are frequently reported in food and feed, being the T-2 toxin (T-2) the mycotoxin which possesses the highest toxicity. According to EFSA, T-2 is found in various cereal grains used in food and feed products, mainly in oats, and it has a high environmental impact due to its mechanisms of toxicity. However, recent information on its genotoxic and mutagenic effects is lacking. This work aimed to evaluate the genotoxic and mutagenic potential of T-2 in vitro. For this purpose, HepG2 cells were exposed to 15, 30, and 60 nM T-2 for 24 h, then the DNA damage was evaluated by the micronucleus and the comet assays. In addition, point mutation analysis was performed by the bacterial reverse mutation test using 0.15-60 nM of T-2 concentrations. The results showed chromosomal damage at 60 nM T-2 since significantly more MN appeared at this concentration than in the control samples. Regarding the comet assay, DNA double helix breaks appeared at all concentrations tested and, in a concentration-dependent manner. However, no mutagenic effects were observed at any of the concentrations tested for the Salmonella typhimurium (S. Typhimurium) strains TA98, TA100, TA1535, TA1537, or the Escherichia coli (E. Coli) WP2 strain in the absence or presence of a metabolic activation system. Therefore, these results showed that T-2 mycotoxin produced genotoxic effects by MN and comet assay, while no mutagenicity was observed. However, further research simulating different metabolic activation pathways and the combined exposure of this mycotoxin with other mutagenic chemicals that could be present in the diet is necessary to discard the mutagenic potential of T-2 fully. These results highlight the carcinogenic potential and danger associated with T-2 exposure and should be considered to prevent associated food risks for the human population.

Evaluation of the Bioaccessible Fraction of T-2 Toxin from Cereals and Its Effect on the Viability of Caco-2 Cells Exposed to Tyrosol.

The bioaccessibility of mycotoxins is an important factor that has to be considered when assessing the risk they pose to human health. Bioactive compounds like phenolics could play a protective role against the toxic effects of contaminants. In this work, the bioaccessible fraction of the T-2 toxin (T-2) contained in breakfast cereals and its effect on the viability of Caco-2 cells were investigated. Furthermore, the effect of tyrosol (a polyphenol abundant in EVOO) on T-2-induced cytotoxicity was evaluated in the same cell line. After standardized in vitro gastrointestinal digestion, the T-2 toxin was released from T-2-spiked breakfast cereals and further quantified by UHPLC-MS/MS. The bioaccessible fraction of T-2 was 51 ± 4%. The cell viability study was performed by pre-treating the cells for 24 h with tyrosol (25, 50 and 100 µM) and subsequently adding T-2 at 15 nM or by treating the cells with a combination of tyrosol and T-2. In the simultaneous treatment, 25 µM tyrosol prevented the toxic effects produced by the exposure to T-2 at 15 nM; however, cytotoxic effects were observed for the other combinations tested. The pre-treatment of Caco-2 cells with tyrosol did not attenuate the cytotoxic effects caused by exposure to T-2. These results suggest that tyrosol at low concentrations (25 µM) could exert a cytoprotective effect on Caco-2 cells against 15 nM T-2 when administered simultaneously with T-2. However, more studies are required to corroborate this hypothesis.

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.

Enhancement of the Antioxidant Effect of Natural Products on the Proliferation of Caco-2 Cells Produced by Fish Protein Hydrolysates and Collagen.

A large amount of fish side streams are produced each year, promoting huge economic and environmental problems. In order to address this issue, a potential alternative is to isolate the high-added-value compounds with beneficial properties on human health. The objectives of this study were to determine the effect of hydrolyzed fish protein and collagen samples on cell proliferation, as well as to determine the specific influence of minerals and metals on this effect and whether dietary antioxidants can enhance cell proliferation. The results of hydrolyzed fish protein and collagen samples showed negative effects on Caco-2 cell proliferation at the highest concentrations tested. Moreover, the pre-treatment of these hydrolyzates with vitamin C and E, quercetin and resveratrol increased the proliferation of bioaccessible fractions of hydrolyzated fish protein and collagen samples compared to the bioaccessible fractions without pre-treatment. The highest mineral concentrations were found for P, Ca and Mg. The metals found in the pure hydrolyzates were As, Cd, Hg and Pb; however, they appeared at almost undetectable levels in bioavailable fractions. It can be concluded that the consumption of hydrolyzates of fish by-products is an interesting strategy for complying with EFSA recommendations regarding fish consumption while at the same time reducing fish waste.

Amitraz and Its Metabolites: Oxidative Stress-Mediated Cytotoxicity in HepG2 Cells and Study of Their Stability and Characterization in Honey.

The population decrease of bees that has been observed in recent years due to the Varroa destructor parasite may endanger the production of bee-products whose demand is on the rise. To minimize the negative effects caused by this parasite, the pesticide amitraz is commonly used by beekeepers. Based on these, the objectives of this work are to determine the toxic effects caused by amitraz and its metabolites in HepG2 cells, as well as its determination in honey samples and the study of its stability with different heat treatments commonly used in the honey industry and its relationship with the amount of 5-hydroxymethylfurfural (HMF) produced. Amitraz significantly decreased cell viability by MTT assay and total protein content (PC) assay, being more cytotoxic than its metabolites. Amitraz and its metabolites caused oxidative stress by Lipid Peroxidation (LPO) production and Reactive Oxygen Species (ROS) generation. Residues of amitraz and/or its metabolites were found in analyzed honey samples, with 2,4-Dimethylaniline (2,4-DMA) being the main metabolite confirmed by high-performance liquid chromatography-high resolution mass spectrometry (HPLC-QTOF HRMS). Amitraz and its metabolites resulted as unstable even at moderate heat treatments. Additionally, a positive correlation in terms of HMF concentration in samples and the severity of heat treatment was also observed. However, quantified amitraz and HMF were within the levels set in the regulation.

In Vitro and Predictive Computational Toxicology Methods for the Neurotoxic Pesticide Amitraz and Its Metabolites.

The Varroa destructor parasite is responsible for varroasis in honeybees worldwide, the most destructive disease among parasitic diseases. Thus, different insecticides/acaricides have been widely used within beehives to control these parasitic diseases. Namely, amitraz is the most used acaricide due to its high efficacy shown against Varroa destructor. However, pesticides used for beehive treatments could be incorporated into the honey and accumulate in other hive products. Hence, honeybee health and the impairment of the quality of honey caused by pesticides have gained more attention. Amitraz and its main metabolites, N-(2,4-dimethylphenyl) formamide (2,4-DMF) and 2,4-dimethylaniline (2,4-DMA), are known to be potent neurotoxicants. In this research, the cytotoxicity of amitraz and its metabolites has been assessed by MTT and PC assays in HepG2 cells. In addition, possible target receptors by in silico strategies have been surveyed. Results showed that amitraz was more cytotoxic than its metabolites. According to the in silico ADMEt assays, amitraz and its metabolites were predicted to be compounds that are able to pass the blood-brain barrier (BBB) and induce toxicity in the central and peripheral nervous systems. The main target class predicted for amitraz was the family of A G protein-coupled receptors that comprises responses to hormones and neurotransmitters. This affects, among other things, reproduction, development, locomotion, and feeding. Furthermore, amitraz and its metabolites were predicted as active compounds interacting with diverse receptors of the Tox21-nuclear receptor signaling and stress response pathways.

Evaluation of cytotoxicity, analysis of metals and cumulative risk assessment in microalgae.

Microalgae are one promising source for the production of bioactive compounds. However, microalgae can accumulate harmful substances. So, our objectives were (i) to evaluate cell viability after Phaeodactylum triconutum (0% and 65% cell disruption, DR) and Tetraselmis chuii (0% and 67% DR) freeze-dried exposure in HepG2 cells by MTT assay; (ii) to evaluate cell viability after P. triconutum and T. chuii extract exposure; (iii) to assess the effect in cell viability when they were simultaneously exposed to T-2 toxin and, (iv) to evaluate if inflammatory response is related to the mechanism of toxicity of these microalgae by qPCR assays. Results demonstrated that cell viability did not increase after freeze-dried microalgae exposure in HepG2 cells. And, no IC50 values were observed. However, an increase in HepG2 cell viability after exposure of T. chuii 0% DR extract at 5, 25 and 100 µg/mL was observed. Additionally, 1:64 diluted T. chuii 0% DR with IC50/4 T-2 and with IC50/2 T-2 and 1:32 diluted T. chuii 0% DR with IC50/4 T-2 showed an increase in cell viability. Both microalgae increased the relative TNF-α, IL-1ß and IL-6 mRNA expression. Concluding, no cytotoxic effect was evidenced but, it was noted up-regulation of inflammatory genes after T. chuii exposure in HepG2 cell. Thus, more studies related the mechanistic toxicity of microalgae are needed to evaluate the potential toxicological risk of inflammation of these novel foods. .

Stressful Effects of T-2 Metabolites and Defense Capability of HepG2 Cells.

The T-2 toxin (T-2), a mycotoxin produced by several species of Fusarium which belongs to group A of trichothecenes, is rapidly metabolized, and its main metabolites are HT-2, Neosolaniol (Neo), T2-triol and T2-tetraol. In this work, the antioxidant defense system of HepG2 cells against oxidative stress induced by T-2 and its metabolites was evaluated. The results obtained demonstrated that there is an overall decrease in glutathione (GSH) levels after all mycotoxins exposure. Moreover, the GSH levels and the enzymatic activities related to GSH (GPx and GST) increased with NAC pre-treatment (glutathione precursor) and decreased with BSO pre-treatment (glutathione inhibitor). The GPx activity is increased by T2-tetraol. The GST activity increased after T-2 and T2-triol exposure; however, T2-tetraol decreased its activity. Furthermore, CAT activity increased after T-2 and T2-triol; nevertheless, Neo decreased its activity. Finally, SOD activity is increased by all mycotoxins, except after T-2 exposure. So, the damage associated with oxidative stress by T-2 and its metabolites is relieved by the antioxidant enzymes system on HepG2 cells.

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).

Effect of Phenolic Extract from Red Beans (Phaseolus vulgaris L.) on T-2 Toxin-Induced Cytotoxicity in HepG2 Cells.

Red beans contain human bioactive compounds such as polyphenols. Several in vitro studies have proposed the natural compounds as an innovative strategy to modify the toxic effects produced by mycotoxins. Hence, in this work, a complete investigation of the polyphenolic fraction of red beans was performed using a Q-Orbitrap high-resolution mass spectrometry analysis. Notably, epicatechin and delphinidin were the most detected polyphenols found in red bean extracts (3.297 and 3.108 mg/Kg, respectively). Moreover, the red bean extract was evaluated against the T-2 toxin (T-2) induced cytotoxicity in hepatocarcinoma cells (HepG2) by direct treatment, simultaneous treatment, and pre-treatment assays. These data showed that T-2 affected the cell viability in a dose-dependent manner, as well as observing a cytotoxic effect and a significant increase in ROS production at 30 nM. The simultaneous treatment and the pre-treatment of HepG2 cells with red bean extract was not able to modify the cytotoxic T-2 effect. However, the simultaneous treatment of T-2 at 7.5 nM with the red bean extract showed a significant decrease in ROS production, with respect to the control. These results suggest that the red bean extract could modulate oxidative stress on HepG2 cells.

Cytoprotective Effects of Fish Protein Hydrolysates against H2O2-Induced Oxidative Stress and Mycotoxins in Caco-2/TC7 Cells.

Many studies report the potent antioxidant capacity for fish protein hydrolysates, including radical scavenging activity and inhibition ability on lipid peroxidation (LPO). In this study, the in vitro cytotoxicity of protein hydrolysates from different salmon, mackerel, and herring side streams fractions was evaluated in the concentration range from 1 to 1:32 dilution, using cloned human colon adenocarcinoma cells TC7 (Caco-2/TC7) by MTT and PT assays. The protein hydrolysates' antioxidant capacity and oxidative stress effects were evaluated by LPO and reactive oxygen species (ROS) generation, respectively. The antioxidant capacity for pure and bioavailable hydrolysate fraction was also evaluated and compared. Additionally, mycotoxin levels were determined in the fish protein hydrolysates, and their cytoprotective effect against T-2 toxin was evaluated. Both hydrolysates and their bioavailable fraction induced similar cell viability rates. The highest cytoprotective effect was obtained for the salmon viscera protein hydrolysate (HSV), which increased the cell viability by 51.2%. ROS accumulation induced by H2O2 and LPO was suppressed by all pure hydrolysates. The cytoprotective effect of hydrolysates was observed against T-2. Moreover, the different fish fraction protein hydrolysates contain variable nutrients and unique bioactive peptide composition showing variable bioactivity, which could be a useful tool in developing dietary supplements with different target functional properties.

Interactions between T-2 toxin and its metabolites in HepG2 cells and in silico approach.

The T-2 toxin (T-2) is commonly metabolized to HT-2 toxin (HT-2), Neosolaniol (NEO), T2-triol and T2-tetraol and they can modify the toxicity of T-2. In this study, T-2 and its modified forms were evaluated by in vitro and in silico methods. The in vitro cytotoxicity individually was evaluated by MTT and Total Protein Content (PC) assays in human hepatocarcinoma (HepG2) cells. The order of IC50 was T-2 tetraol > T-2 triol > NEO > T-2 = HT-2. The T-2 and HT-2 evidenced the highest cytotoxic effect in HepG2 cells individually. No differences were observed in binary combinations tested and the two mycotoxins in the mixture tested individually. The T-2+HT-2 combination showed the highest toxic potential with the lowest IC50 value of 34.42 ± 0.58 nM at 24 h. All binary combinations exhibited antagonistic interactions. The ADME and toxicity profile of mycotoxins were obtained by the in silico admetSAR predictive model which determines the metabolic and toxicological approaches in order to know if these mycotoxins might be taken into consideration to support a more realistic and adequate risk assessment.

Improved Extraction Efficiency of Antioxidant Bioactive Compounds from Tetraselmis chuii and Phaedoactylum tricornutum Using Pulsed Electric Fields.

Pulsed electric fields (PEF) is a promising technology that allows the selective extraction of high-added value compounds by electroporation. Thus, PEF provides numerous opportunities for the energy efficient isolation of valuable microalgal bioactive substances (i.e., pigments and polyphenols). The efficiency of PEF-assisted extraction combined with aqueous or dimethyl sulfoxide (DMSO) solvents in recovering pigments and polyphenols from microalgae Tetraselmis chuii (T. chuii) and Phaeodactylum tricornutum (P. tricornutum) was evaluated. Two PEF treatments were applied: (1 kV/cm/400 pulses, 3 kV/cm/45 pulses), with a specific energy input of 100 kJ/kg. The total antioxidant capacity (TAC) was positively influenced by the use of DMSO. The highest TAC in the T. chuii culture was achieved at a lower extraction time and electric field than for P. tricornutum. The use of DMSO only improved the polyphenols' extraction for P. tricornutum, whereas the PEF and extraction time were more important for T. chuii. Carotenoids and chlorophyll a were more efficiently extracted using DMSO, while chlorophyll b levels were higher following aqueous extraction for both microalgae. In P. tricornutum, the TAC and pigment extraction efficiency were in general higher at lower extraction times. It can be concluded that PEF may be a promising alternative for the enhancement of the selective extraction of antioxidant bioactive compounds from microalgae.

T-2 toxin and its metabolites: Characterization, cytotoxic mechanisms and adaptive cellular response in human hepatocarcinoma (HepG2) cells.

The T-2 toxin (T-2) is a type A trichothecene produced by Fusarium species, and the most cytotoxic mycotoxin of the group. A study was made to determine T-2 cytotoxicity in human hepatocarcinoma (HepG2) cells; evaluate whether there is an adaptive response of HepG2 cells exposed to low concentrations of T-2; identify the T-2 metabolites by LC-Q-TOF MS; and determine whether T-2 disrupts cell proliferation in HepG2 cells. The IC50 values obtained ranged from 61.9 ± 2.4 nM to 70.7 ± 7.4 nM. No adaptive response was observed. There was no evidence of extra- or intracellular accumulation of T-2 after 24 h of exposure as determined by LC-Q-TOF MS. However, some T-2 metabolites such as HT-2 toxin, neosolaniol and T-2 triol showed important (>75%) intracellular accumulation. Cell distribution was significantly increased in SubG0/G1 phase (11.8-fold higher) and decreased (12%) in G2/M phase at 60 nM T-2, versus the control. Simultaneously, increased necrosis (238%) and apoptosis/necrosis (up to 35.5%) were observed in HepG2 cells exposed to T-2. In conclusion, the results show that T-2 leads to loss of cell viability without an adaptive response, and that the metabolites generated play an important role in T-2 cytotoxicity, increasing HepG2 cell damage.

Does low concentration mycotoxin exposure induce toxicity in HepG2 cells through oxidative stress?

The purpose of this study was to determine whether exposure to low concentrations of deoxynivalenol (DON), T-2 toxin (T-2) and patulin (PAT) in a human hepatocellular carcinoma cell line (HepG2) exerts toxic effects through mechanisms related to oxidative stress, and how cells deal with such exposure. Cell viability was determined by the MTT and protein content (PC) assays over 24, 48 and 72 h. The IC50 values detected ranged from >10 to 2.53 ± 0.21 µM (DON), 0.050 ± 0.025 to 0.034 ± 0.007 µM (T-2) and 2.66 ± 0.66 to 1.17 ± 0.21 µM (PAT). The key players in oxidative stress are the generation of reactive oxygen species (ROS), lipid peroxidation (LPO) and mitochondrial membrane potential (MMP) dysfunction. The results obtained showed that PAT, DON and T-2 did not significantly increase LPO or ROS production with respect to the controls. Moreover, PAT and DON did not alter MMP, though T-2 increased MMP at the higher concentrations tested (17 and 34 nM). In conclusion, the exposure of HepG2 cells to nontoxic concentrations of T-2 condition them against subsequent cellular oxidative conditions induced by even higher concentrations of mycotoxin.

In silico and in vitro prediction of the toxicological effects of individual and combined mycotoxins.

3-Acetyldeoxynivalenol (3-AcDON) and 15-acetyldeoxynivalenol (15-AcDON) are converted to deoxynivalenol (DON) in vivo and their simultaneous presence may increase DON intake. Mixtures of DON and its derivatives are a public health concern. In this study DON, 3-AcDON and 15-AcDON were evaluated in vitro and in silico. The in vitro cytotoxicity of DON and its derivatives individually and combined was determined by the Neutral Red (NR) assay in human hepatocarcinoma (HepG2) cells. The concentrations tested were from 1.25 to 15 µM (DON) and from 0.937 to 7.5 µM (DON derivatives). The IC50 values were from >15 to 2.55 µM (DON), from 1.77 to 1.02 µM (3-AcDON), and from 4.05 to 1.68 µM (15-AcDON). 3-AcDON was the most cytotoxic molecule in HepG2 cells. The concentrations tested in combinations ranged from 0.5625 to 4.5 µM (DON), and from 0.094 to 0.75 µM (DON derivatives), with ratios of 1:6 (DON+3-AcDON and DON+15-AcDON), 1:1 (3-AcDON+15-AcDON) and 1:6:6 (DON+3-AcDON+15-AcDON). The DON+15-AcDON mixture exhibited additive effects, while the rest showed synergistic effects. In silico methods assess individual mycotoxins. Absorption, Distribution, Metabolism, Excretion and Toxicity of mycotoxins were predicted using in silico SwissADME tools. Absorption, Distribution, Metabolism and Excretion profile prediction shows high gastrointestinal absorption and CYP3A4 mediated metabolism.

Micronucleus induction and cell cycle alterations produced by deoxynivalenol and its acetylated derivatives in individual and combined exposure on HepG2 cells.

Mycotoxins are produced by a number of fungal genera spp as e.g. Aspergillus, Penicillium, Alternaria, Fusarium and Claviceps. 3-Acetyl-Deoxynivalenol (3-A-DON) and 15-Acetyl-Deoxynivalenol (15-ADON) which are produced by Fusarium, chemically belong to trichothecenes and occur in significant amounts as modified forms of deoxynivalenol (DON) in various cereal crops and processed grains. This study aims to determine the cytotoxicity, cell cycle and genotoxicity of the mycotoxins DON, 3-A-DON and 15-A-DON on HepG2 cells. Cytotoxic concentration range studied was from 100 to 3.1 µM for DON and 12.5 to 0.04 µM for 3-A-DON and 15-A-DON by the Neutral Red (NR) assay, over 24, 48 and 72 h. Potential of toxicity of 3-ADON in HepG2 cells was the highest at all times assayed. Cell cycle arrest in G0/G1 and G2/M phase was detected for all mycotoxins either in individually or in combined treatment in HepG2 cells. Genotoxicity was performed through micronuclei (MN) induction (TG 487) revealing significant effects for 3-ADON mycotoxin and in several combinations. It was evidenced that cell cycle alterations detected were associated to MN induction at all doses assayed, reaching the highest induction in tertiary combinations and in the binary combination 3-ADON + 15-ADON.