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

[EN] 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.This work is part of a research project funded by the Spanish Ministry of Science and Innovation PID2020-115871RB-I00, MCIN/AEI/10.13039/501100011033. Mercedes Taroncher is grateful for the predoctoral grant PRE2021-096941 provided by the Spanish Ministry of Science and Innovation project. Cristina Fuentes was supported by a Margarita Salas Postdoctoral Fellowship from Universitat Politecnica de Valencia funded by Ministerio de Universidades and the European Union-NextGeneration EU.Taroncher, M.; Fuentes López, C.; Rodríguez-Carrasco, Y.; Ruiz, M. (2024). Assessment of the genotoxic and mutagenic effects induced by T-2 mycotoxin in HepG2 cells. Toxicology (Online). 501. https://doi.org/10.1016/j.tox.2023.15371250

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

Mecanismos de acción tóxica producidos por la toxina T-2 y sus metabolitos en modelos de células hepáticas humanas 2D y 3D y mecanismos de defensa antioxidantes

En la presente Tesis Doctoral se ha llevado a cabo la evaluación de los efectos tóxicos producidos por la toxina T-2 y sus metabolitos HT-2, neosolaniol (Neo), T2-triol y T2-tetraol mediante métodos alternativos a la experimentación animal, concretamente modelos de predicción in silico y modelos in vitro en células de hepatocarcinoma humano (HepG2). Debido a que la T-2 es la micotoxina más citotóxica de los tricotecenos, los resultados obtenidos pueden contribuir a un mayor conocimiento de sus mecanismos de acción toxicológica. De esta manera se puede realizar una evaluación del riesgo más precisa, y adoptar medidas adecuadas para prevenir o mitigar el daño producido tras su exposición. Para ello, se evalúan las propiedades toxicológicas y de los procesos ADME de la T-2, HT-2, Neo, T2-triol y T2-tetraol utilizando los métodos de predicción in silico AdmetSAR y SwissAdme. Tras observarse potenciales efectos tóxicos, se procede a la evaluación de la toxicidad de la T-2 y sus metabolitos HT-2, Neo, T2-triol y T2-tetraol en un modelo in vitro, con células HepG2 de forma individual y combinada, mostrando valores de IC50 entre 59,6 y 2770 nM. A continuación, se identifica y cuantifica la T-2 y sus principales productos de biotransformación en el medio extra e intracelular tras 24 h de exposición de la T-2 a las células HepG2, observándose la completa metabolización de T-2 y la fluctuación de los niveles de sus metabolitos tanto en la fracción celular como en el medio de cultivo, siendo HT-2 el mayoritario. Debido a la coexistencia de la T-2 y sus metabolitos, se determina el tipo de interacción entre ellos, y se evidencian diferentes efectos dependiendo de la combinación de micotoxinas y las concentraciones en la mezcla. Así, se observa efectos antagónicos en las combinaciones de T-2 + T2-triol, T-2 + Neo, Neo + T2-triol, Neo + T2-tretraol y Neo + HT-2, y efectos antagónicos a concentraciones bajas y aditivos a altas en las combinaciones de T-2 + HT-2, T-2 + T2-tetraol, T2-triol + HT-2, T2-triol + T2-tretraol y T2-tetraol + HT-2. Como posible mecanismo de acción tóxica, a continuación se evalúa la capacidad de la T-2 y sus metabolitos de generar especies reactivas de oxígeno (ROS) y el papel protector de los sistemas de defensa celular y enzimático. Respecto a la defensa celular, se observa una disminución general del glutatión (GSH) tras la exposición a las micotoxinas. Sin embargo, se observa una gran variedad de resultados, dependiendo de la micotoxina y de la concentración evaluada, en cuanto a la defensa enzimática (glutatión peroxidasa, GPx; glutatión transferasa, GST), catalasa (CAT) y superóxido dismutasa (SOD) en las células HepG2. El papel del GSH y las enzimas relacionadas (GPx y GST) en la defensa de las células HepG2 expuestas a la T-2 y sus metabolitos se confirma con el incremento de su actividad a nivel celular tras la adición de N-acetil-cisteína (NAC) y la reducción tras la adición de D-L-butionina-(S, R)-sulfoximina (BSO). Por otra parte, también se evalúa el efecto citoprotector de sustancias bioactivas presentes en legumbres (judías rojas), microalgas (Tetraselmis chuii y Phaeodactylum tricornutum) e hidrolizados de pescado, todos ellos componentes de la dieta Mediterránea y debido a su alto contenido en antioxidantes. Por otra parte, la exposición de las células HepG2 a la T-2 pone de manifiesto una alteración de la proliferación celular observada con la disrupción del ciclo celular, que podría estar relacionado con los procesos de muerte celular apoptosis y necrosis. Además, se observa daño al ADN tras la exposición a T-2, el cual se determinó a través de ensayos de genotoxicidad, concretamente el ensayo de micronúcleos y el ensayo del cometa. No obstante, la exposición a T-2 no causó efecto mutagénico mediante el ensayo de la mutación genética reversa bacteriana. Finalmente, se aplica un modelo celular tridimensional (3D) de esferoides de células hepáticas en dispositivos microfluídicos para simular el entorno biológico in vivo y obtener resultados más precisos y relevantes para el estudio de la citotoxicidad de la T-2 en modelos hepáticos. Se estudian y comparan dos modelos de cultivo celular 3D en dispositivos microfluídicos: esferoides de monocultivo con células HepG2 y esferoides de co-cultivo con células HepG2 y LX2. Con estos modelos, se evalúan los efectos citotóxicos de la T-2 mediante diferentes procesos, tales como viabilidad celular, albúmina, enzimas metabólicas hepáticas y citoquinas inflamatorias. Los resultados obtenidos con los esferoides de co-cultivo en dispositivos microfluídicos confirman que los efectos observados tras la exposición de la T-2 presentan un enfoque más realista de las condiciones hepáticas que los obtenidos con esferoides de monocultivo o en placas estándar. En resumen, los resultados obtenidos indican que la T-2 y sus metabolitos podrían suponer un riesgo para la salud humana. Por lo que sería necesario ampliar los conocimientos de los mecanismos de toxicidad para poder prevenir efectos adversos de la T-2 y sus metabolitos y reducir la exposición a través de la dieta para proteger la salud del consumidor.In this Doctoral Thesis, the evaluation of the toxic effects produced by the T-2 toxin and its metabolites HT-2, neosolaniol (Neo), T2-triol and T2-tetraol has been carried out using alternative methods to animal testing. Specifically, in silico predictive models and in vitro models using human hepatocellular carcinoma (HepG2) cells. Since T-2 is the most cytotoxic mycotoxin of the trichothecenes, the results obtained may contribute to a better understanding of its toxicological mechanisms of action. This will allow a more accurate risk assessment to be made and appropriate measures to be taken to prevent or mitigate harm following exposure. For this purpose, the toxicological properties and ADME processes of T-2, HT-2, Neo, T2-triol and T2-tetraol were evaluated using the in silico predictive methods AdmetSAR and SwissAdme. After potential toxic effects were observed, the toxicity of T-2 and its metabolites HT-2, Neo, T2-triol and T2-tetraol was evaluated in an in vitro model using HepG2 cells individually and in combination. IC50 values ranging from 59.6 to 2770 nM were obtained. Next, T-2 and its main biotransformation products were identified and quantified in the extra- and intracellular medium after 24 h of T-2 exposure to HepG2 cells, showing complete metabolization of T-2 fluctuating levels of its metabolites both in the cellular fraction and in the culture medium, with HT-2 being the major one. Due to the coexistence of T-2 and its metabolites, the type of interaction between them was determined and different effects were found depending on the combination of mycotoxins and the concentrations in the mixture. Thus, antagonistic effects are observed for combinations of T-2 + T2-triol, T-2 + Neo, Neo + T2-triol, Neo + T2-tretraol and Neo + HT-2, and both antagonistic effects at low and additive effects at high concentrations for combinations of T-2 + HT-2, T-2 + T2-tetraol, T2-triol + HT-2, T2-triol + T2-tretraol and T2-tetraol + HT-2. As a possible mechanism of toxicological action, the ability of T-2 and its metabolites to generate reactive oxygen species (ROS) and the protective role of cellular and enzymatic defense systems were evaluated. In terms of cellular defense, a general decrease in glutathione (GSH) was observed after exposure to mycotoxins. However, depending on the mycotoxin and the concentration tested, a wide range of results were observed in terms of enzymatic defense (glutathione peroxidase, GPx; glutathione transferase, GST), catalase (CAT) and superoxide dismutase (SOD) in HepG2 cells. The role of GSH and related enzymes (GPx and GST) in the defense of HepG2 cells exposed to T-2 and its metabolites was confirmed by the increase in their activity at the cellular level after the addition of N-acetyl-cysteine (NAC) and the decrease after the addition of D-L-buthionine-(S, R)-sulfoximine (BSO). The cytoprotective effect of bioactive substances present in legumes (red beans), microalgae (Tetraselmis chuii and Phaeodactylum tricornutum) and fish hydrolysates, all of which are components of the Mediterranean diet and are known their high antioxidant content, was also evaluated. Moreover, exposure of HepG2 cells to T-2 revealed an alteration in cell proliferation with cell cycle disruption, which could be related to the processes of cell death, apoptosis and necrosis. In addition, DNA damage was observed after T-2 exposure as determined by genotoxicity assays, namely the micronucleus assay and the comet assay. However, exposure to T-2 did not induce mutagenic effects using the bacterial reverse gene mutation assay. Finally, a three-dimensional (3D) cellular model of liver cell spheroids was used in microfluidic devices to simulate the in vivo biological environment and to obtain more accurate and relevant results for the study of T-2 cytotoxicity in liver models. Two 3D cell culture models were investigated and compared in microfluidic devices: monoculture spheroids with HepG2 cells and co-culture spheroids with HepG2 and LX2 cells. The models were used to assess the cytotoxic effects of T-2 by different measures, such as cell viability, albumin, hepatic metabolic enzymes and inflammatory cytokines. The results obtained with co-culture spheroids in microfluidic devices confirmed that the effects observed after T-2 exposure represent a more realistic approach to liver conditions than those obtained with monoculture spheroids or in standard plates. In conclusion, the results obtained indicate that T-2 and its metabolites could represent a risk to human health. Therefore, further knowledge of the mechanisms of toxicity would be necessary to prevent adverse effects of T-2 and its metabolites and to reduce dietary exposure in order to protect consumer health.Contrato predoctoral para la formación de doctores de la convocatoria 2021, FPI, Ministerio de Ciencia e Innovació

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

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 microalgaepublishedVersio

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

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

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

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

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)