M.J. Ruiz

Reactive oxygen species induced by beauvericin, patulin and zearalenone in CHO-K1 cells.

The cytotoxic effects of mycotoxins, induction of reactive oxygen species (ROS) and generation of lipid peroxidation products in CHO-K1 cells were determined as function of increasing time of exposure and concentrations of beauvericin (BEA), patulin (PAT) and zearalenone (ZEA). The end points were evaluated after 24h of exposure, by the tetrazolium salt (MTT) and neutral red (NR) assays. The IC(50) values obtained on the MTT and NR assays ranged from 0.69 to 79.40 microM and 4.40 to 108.76 microM, respectively. To determine the intracellular production of ROS, the intensity of fluorescence emitted from the probe H(2)-DCFDA was measured. The relative intensity of fluorescence from cells incubated with BEA, PAT and ZEA was approximately 4-, 7- and 4-fold higher in comparison with control cells at 0 min, respectively. Lipid peroxidation induced by ROS generation was assessed using the thiobarbituric acid reactive substances (TBARS) method for 2, 24 and 48 h. The malondialdehyde (MDA) production was increased with BEA and PAT exposure in a concentration- and time-dependent manner. MDA was not increased after 1 and 5 microM ZEA exposures for 2h, but was slightly increased at 50 microM. In conclusion, PAT was the most cytotoxic mycotoxin to CHO-K1 cells, followed by BEA and ZEA. Mycotoxins reduce cell viability correlated with the increases of ROS generation and MDA formation in concentration- and time-dependent manner. These data suggested that cytotoxicity and ROS generation are mechanisms of mycotoxins mediated toxicity.

Dietary Administration of High Doses of Pterostilbene and Quercetin to Mice Is Not Toxic

The aim of this study is to evaluate possible harmful effects of high doses of t-pterostilbene (t-PTER) and quercetin (QUER) in Swiss mice. Mice were fed during 28 days at doses of 0, 30, 300, and 3000 mg/kg body weight/day of t-PTER, QUER, or a mixture of both, t-PTER + QUER, which are equivalent to 5, 50, and 500 times, respectively, the estimated mean human intake of these polyphenols (25 mg/day). Daily oral administration of QUER, t-PTER, or a mixture of both of them did not cause mortality during the experimental period. There were no differences in food and water consumption on sex. No significant body weight gain in the male or female groups was observed. Red blood cell number and the hematocrit increased after polyphenols administration compared to control groups. Biochemical parameters were not affected. Histopathological examination revealed no alterations in clinical signs or organ weight at any dose.

Co-occurrence and risk assessment of mycotoxins in food and diet from Mediterranean area.

The contents of 14 mycotoxins were studied in samples of different cereals and cereal products from four countries of the Mediterranean region. Two hundred and sixty-five samples from Spain, Italy, Morocco and Tunisia were analysed. Samples were extracted with matrix solid-phase dispersion (MSPD) and determined by liquid chromatography-tandem mass spectrometry with a triple quadrupole mass analyser. The percentage of total samples contaminated was 53%. The frequency of contaminated samples from Spain, Italy, Tunisia and Morocco was 33%, 52%, 96% and 50%, respectively. Nivalenol and beauvericin were the most predominant mycotoxins. This is the first international report to study the presence of several mycotoxins in different types of cereal (rice, wheat, maize, rye, barley, oat, spelt and sorghum) and cereal products (snacks, pasta, soup, biscuits and flour) from the Mediterranean area, estimate the intake of mycotoxins and evaluate the risk assessment.

Cytotoxic effects of mycotoxin combinations in mammalian kidney cells

The cytotoxicity of three Fusarium mycotoxins (beauvericin, deoxynivalenol and T-2 toxin) has been investigated using the NR assay, after 24, 48 and 72h of incubation. The IC(50) values ranged from 6.77 to 11.08, 3.30 to 10.00 and 0.004 to 0.005 for beauvericin, deoxynivalenol and T-2 toxin, respectively. Once the potential interaction has been detected, a quantitative assessment is necessary to ensure and characterize these interactions, that is, each mycotoxin contributes to the toxic effect in accord with its own potency. Combination of mycotoxins was determined in Vero cells after 24, 48 and 72h of exposure. Isobolograms and median effect method of Chou and Talalay were used to assess the nature and quantitative aspects of interaction observed between studied mycotoxins. Median effect analysis was used to calculate the combination index (CI) with values >1 indicating synergism, 1 additive effect, and <1 antagonism. CI values of BEA+DON (1.22-2.74), BEA+T-2 toxin (1.43-5.89), DON+T-2 toxin (3.13-7.62) and BEA+DON+T-2 toxin (1.32-2.68) for 24, 48 and 72h produced antagonistic effects in Vero cells. The highest antagonistic effect in Vero cells was observed with binary DON and T-2 toxin mixture.

Toxicological interactions between the mycotoxins beauvericin, deoxynivalenol and T-2 toxin in CHO-K1 cells in vitro

Beauvericin (BEA), deoxynivalenol (DON) and T-2 toxin (T-2) are important food-borne mycotoxins that have been implicated in human health. In this study, the acute toxicity of individual and combined mycotoxins (BEA, DON and T-2) were tested in immortalized hamster ovarian cells (CHO-K1) at 24, 48 and 72 h of exposure, by the tetrazolium salt (MTT) and neutral red (NR) assays. The IC(50) values obtained for all mycotoxins by the MTT and NR assays ranged from 0.017 to 12.08 μM and from 0.042 to 17.22 μM, respectively. Both, individual and combined mycotoxins demonstrated a significant cytotoxic effect in CHO-K1 cells in a dose-dependent manner. When mycotoxins were assayed individually, T-2 showed the strongest IC(50) values (from 0.017 to 0.052 μM), by both endpoints tested, followed by DON (0.53-2.30 μM) and BEA, showing this last one, the weakest IC(50) values (from 2.77 to 17.22 μM). On the other hand, cytotoxicity interactions were evaluated by the isobologram method. In acute binary tests, DON+BEA (CI=1.60-25.07) and DON+T-2 (CI=1.74-7.71) showed antagonism at 24, 48 and 72 h of exposure. By contrast, the binary BEA+T-2 combination (CI=0.35-0.78) showed synergism at all time of exposure tested. The tertiary BEA+DON+T-2 combination demonstrated synergism effect (CI=0.47-0.86) after 24 and 48 h of exposure; however moderate antagonistic effect (CI=1.14-1.60) was presented after 72 h of exposure at the lower doses. These results provide quantitative evidence regarding potentially important interactions between BEA, DON and T-2 depending of the time of exposure. The combination index-isobologram equation method can serve as a useful tool in food risk assessment. Due to the potent toxic effects of BEA, DON and T-2, its combined exposure might be an important trigger for development of several diseases in humans, from the mycotoxicological point of view, especially after long period of exposure time.

Comparative cytotoxicity study of enniatins A, A1, A2, B, B1, B4 and J3 on Caco-2 cells, Hep-G2 and HT-29

Enniatins (ENs) are ionophoric, phytotoxic, antihelminthic, and antibiotic compounds of hexadepsipeptidic structure produced by several strains of Fusarium spp. The cytotoxicity effect of the ENs A, A(1), A(2), B, B(1), B(4) and J(3) was compared on three tumor cell lines, the human epithelial colorectal adenocarcinoma (Caco-2), the human colon carcinoma (HT-29), and the human liver carcinoma (Hep-G2). The endpoint evaluated was the mitochondrial integrity by using the MTT assays, after 24 and 48 h of incubation. The IC(50) value for EN A(2) on Caco-2 cells, after 24h exposure, was 18.7 ± 4.5 μM and decrease to 2.6 ± 0.7 μM at 48 h of incubation. However, ENs A, A(1), B(1) and B(4) exert pronounced cytotoxic effects in all the cell lines tested by the MTT assay after 24 and 48 h of incubation. The EN A(1) demonstrated to be the most cytotoxic ENs tested. Moreover, no statistical differences were found between the IC(50) values obtained for EN A(1) on Caco-2, HT-29 and Hep-G2, with IC(50) values ranging from 9.1 ± 2.2 μM to 12.3 ± 4.3 μM at 24h and decreasing in a range variable from 1.4 ± 0.7 μM to 2.7 ± 0.8 μM at 48 h. On the other hand, EN A, B(1) and B(4) showed lower cytotoxicity, but in a similar range as the IC(50) values reported on HT-29 (IC(50) values (24h): 16.8 ± 4.3-26.2 ± 6.7 μM), Caco-2 (IC(50) values (24h): 19.5 ± 4.1 μM) and Hep-G2 (IC(50) values (24h): 23.4 ± 5.6-26.2 ± 7.6 μM) cells. Cytotoxic effect with a 48 h of incubation revealed also a significant toxicity of ENs A (IC(50) values ranged from 8.2 ± 1.8 to 11.4 ± 4.6 μM), B(1) (IC(50) values variables from 3.7 ± 0.7 to 11.5 ± 5.3 μM) and B(4) (IC(50) of 4.5 ± 2.9-15.0 ± 4.0 μM). In summary, this study demonstrated that ENs can exert toxic activity at low micromolar concentrations in mammalian cells.

Beauvericin-induced cytotoxicity via ROS production and mitochondrial damage in Caco-2 cells

The cytotoxicity of beauvericin (BEA) on human colon adenocarcinoma (Caco-2) cells was studied as a function of time. Moreover, the oxidative damage and cell death endpoints were monitored after 24, 48 and 72 h. After BEA exposure, the IC₅₀ values ranged from 1.9 ± 0.7 to 20.6 ± 6.9 μM. A decrease in reduced glutathione (GSH; 31%) levels, as well as an increase in oxidized glutathione (GSSG, 20%) was observed. In the presence of BEA, reactive oxygen species (ROS) level was highly increased at an early stage with the highest production of 2.0-fold higher than the control that was observed at 120 min. BEA induced cell death by mitochondria-dependent apoptotic process with loss of the mitochondrial membrane potential (ΔΨm; 9% compared to the control), increase in LPO level (from 120% to 207% compared to the control) and reduced G0/G1 phase, with an arrest in G2/M, in a dose and time-dependent manner. Cell proliferation, apoptosis and ΔΨm determined, were in a dose- time-dependent manner. Moreover, DNA damage was observed after 12.0 μM concentration. This study demonstrated that oxidative stress is one of the mechanism involved in BEA toxicity, moreover apoptosis induction and loss of ΔΨm contribute to its cytotoxicity in Caco-2 cells.

Interactive effects of zearalenone and its metabolites on cytotoxicity and metabolization in ovarian CHO-K1 cells.

Zearalenone (ZEA) is a non-steroidal estrogen mycotoxin with high binding affinity to estrogen receptors. ZEA is rapidly absorbed and metabolized in vivo to α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL). So, mixtures of them may be present in biological systems and suppose a hazard to animals and human health. The aims of this study were to determine the cytotoxic effects of ZEA and its metabolites, alone and in combination in ovarian (CHO-K1) cells during 24, 48 and 72h by the MTT assay; and to investigate the metabolism of the CHO-K1 cells on ZEA, and its conversion into α-ZOL and β-ZOL by CHO-K1 cell after 24 and 48h of exposure. The IC50 value obtained for individual mycotoxins range from 60.3 to >100.0μM, from 30.0 to 33.0μM and from 55.0 to >75.0μM for ZEA, α-ZOL and β-ZOL, respectively. Cytotoxic interactions were assayed by the isobologram method, which provides a combination index (CI) value as a quantitative measure of the degree of the three mycotoxin interaction. The CI values for binary combinations ranged from 0.56±0.15 (synergism at low concentrations) to 5.25±5.10 (addition at high concentrations) and tertiary combinations from 2.95±0.75 (antagonism at low concentrations) to 0.41±0.23 (synergism at high concentrations). The concentration of ZEA and its metabolites was determined with liquid chromatography coupled to the mass spectrometer detector-linear ion trap (LC-MS-LIT). The percentage of ZEA degradation ranged from 4% (24h) to 81% (48h). In the same conditions, α-ZOL and β-ZOL concentration decreased from 8% to 85%. No conversion of ZEA in α-ZOL and β-ZOL was detected. However, at 24h of exposure other degradation products of ZEA and its derived were detected.

Involvement of enniatins-induced cytotoxicity in human HepG2 cells.

Enniatins (ENNs) are mycotoxins found in Fusarium fungi and they appear in nature as mixtures of cyclic depsipeptides. The ability to form ionophores in the cell membrane is related to their cytotoxicity. Changes in ion distribution between inner and outer phases of the mitochondria affect to their metabolism, proton gradient, and chemiosmotic coupling, so a mitochondrial toxicity analysis of enniatins is highly recommended because they host the homeostasis required for cellular survival. Two ENNs, ENN A and ENN B on hepatocarcinoma cells (HepG2) at 1.5 and 3 μM and three exposure times (24, 48 and 72 h) were studied. Flow cytometry was used to examine their effects on cell proliferation, to characterize at which phase of the cell cycle progression the cells were blocked and to study the role of the mitochondrial in ENNs-induced apoptosis. In conclusion, apoptosis induction on HepG2 cells allowed to compare cytotoxic effects caused by both ENNs, A and B. It is reported the possible mechanism observed in MMP changes, cell cycle analysis and apoptosis/necrosis, identifying ENN B more toxic than ENN A.

Presence of Ochratoxin A (OTA) Mycotoxin in Alcoholic Drinks from Southern European Countries: Wine and Beer

The main filamentous fungi producers of mycotoxins are Aspergillus spp., Penicillium spp., and Fusarium spp. Their effect can provoke a broad range of toxic properties including carcinogenicity and neurotoxicity, as well as reproductive and developmental toxicities. Ochratoxin A (OTA) is produced by Aspergillus and Penicillium spp. The purpose of this review was to evaluate the risk assessment of OTA in alcoholic drinks (beer and wine) by compiling the results obtained from studies and reviews related to the presence of OTA in these two drinks from southern European countries in the period 2005-2013 and comparing those results with the legislation available in the European Union.