Fiorenza Minervini

Toxicity and apoptosis induced by the mycotoxins nivalenol, deoxynivalenol and fumonisin B1 in a human erythroleukemia cell line

The toxicity of the mycotoxins nivalenol (NIV), deoxynivalenol (DON) and fumonisin B1 (FB1) were studied in the K562 human erythroleukemia cell line using the Trypan Blue, MTT and BrdU uptake analyses of cytotoxicity, cell metabolism and cell proliferation, respectively. Nuclear staining with propidium iodide and DNA analysis by flow cytometry were used to identify apoptosis and cell cycle distribution. By the MTT and BrdU tests, both NIV and DON were significantly more toxic than FB1 by at least one order of magnitude, with ID50s ranging from 0.5 microM for NIV to 70 microM for FB1. The MTT test indicated that NIV was significantly (approximately four times) more toxic than DON. In contrast, the Trypan Blue test did not reveal any effects of mycotoxin exposure suggesting that, at the concentrations tested, NIV, DON and FB1 did not induce cytotoxicity through plasma membrane damage. Cell cycle analysis suggested apoptotic cytotoxicity, revealing 100% cellular debris at the highest concentrations of NIV and DON and approximately 2.9 times more debris than control at the highest FB1 concentration. Morphological evidence of apoptosis was related to the toxicity of the substances, such that the more toxic NIV and DON resulted in more late stage apoptotic events than FB1. This study suggests that human blood cells are sensitive to mycotoxin exposure, that NIV is more toxic than DON which is more toxic than FB1, and that DNA damage and apoptosis rather than plasma membrane damage and necrosis may be responsible for the observed cytotoxicity.

Zearalenone and reproductive function in farm animals.

Farm animals are exposed to zearalenone through the feed because of the widespread occurrence of this mycotoxin in cereals and clinical reproductive disorders due to mycotoxin effects are often reported in farm animal species. This review describes the metabolism, the mechanistic aspects, the clinical reproductive symptoms and the in vitro effects on functional parameters of oocytes and sperm cells induced by zearalenone and its derivatives in farm animals. The studies on in vitro effects allow to understand the action mechanisms of mycotoxins and, sometime, to explain the in vivo symptoms. The impairment of semen quality and female reproductive function induced by zearalenone could be a factor responsible for the reproductive failure in farm animals.

Toxic effects of the mycotoxin zearalenone and its derivatives on in vitro maturation of bovine oocytes and 17β-estradiol levels in mural granulosa cell cultures

Moulds parasites of livestock foodstuffs alter the quality of grains by synthesizing mycotoxins. Zearalenone (ZEA) and its derivatives (alpha- and beta-zearalenol, zeranol, taleranol and zearalanone) are produced by fungi of the genus Fusarium and, after ingestion via contaminated cereals, may lead to fertility disturbances and other reproductive pathologies. Zearalenone, alpha-zearalenol and zearalanone were tested, at levels ranging from 0.3 to 30 microg/ml, in order to evaluate the effect on the in vitro maturation (IVM) rate of bovine oocytes and on the formation of 17 beta-estradiol in supernatants of mural granulosa cells (GC) cultures. These compounds induced dose-dependent oocyte maturation delay and chromatin abnormalities. Maturation of oocytes to metaphase II (M II) was inhibited in oocytes cultured in the presence of 30 microg/ml ZEA, alpha-zearalenol or zearalanone, with a significant increase in chromatin abnormalities occurring in the presence of ZEA (P<0.05) and alpha-zearalenol (P< 0.001). In preliminary trials on 17 beta-estradiol formation, at the same testing concentration, higher levels of 17 beta-estradiol were found in the presence of alpha-zearalenol (mean value 1.6 ng/ml) with respect to ZEA and zearalanone (mean estradiol concentrations of 0.06 and 0.5 ng/ml, respectively). These data demonstrate a negative effect of ZEA and its derivatives on meiotic progression of bovine oocytes, possibly attributable to a toxic mechanism not related to the binding affinity of these compounds to estrogen receptor sites, and support previous observations that alpha-zearalenol acts as a stronger estrogenic inducer than the original molecule (ZEA).

Toxic effects induced by mycotoxin fumonisin B1 on equine spermatozoa: assessment of viability, sperm chromatin structure stability, ROS production and motility.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm motility by CASA system. Fumonisin B1 did not affect viability of fresh spermatozoa after 2h exposure up to 25 μM. Damage on sperm chromatin structure was observed only in one frozen sample after exposure up to 2.5 x 10⁻⁵ μM FB1 without associated increase of ROS. Increase of ROS, at FB1 levels up to 2.5 x 10⁻⁴ μM, was found on another frozen-thawed sperm sample, may be as a consequence of seminal plasma removal. At 7.5 and 15 μM, FB1 induced reduction of total and progressive motility.

High degradation and no bioavailability of artichoke miRNAs assessed using an in vitro digestion/Caco-2 cell model

Although the cross-kingdom transfer of vegetable miRNAs (miRNAs) in mammalian species, including humans, is still controversial, recent studies have rejected this theory. Based on these recent studies, we hypothesized that artichoke-derived miRNAs (cca-miRNAs) are not adsorbed into human intestinal cells after cooking and in vitro digestion. In order to test this hypothesis, we evaluated miRNA (cca-miRNAs) in the edible part of globe artichokes (head portion), after cooking and digestion by an in vitro digestion system. The cca-miRNA levels were analyzed by real-time PCR (RT-qPCR), and those that withstood cooking and digestion conditions were further analyzed for their bioavailability using an in vitro system (Caco-2/TC7 cell clone). We detected 20 cca-miRNAs after cooking, 5 of which were statistically down-regulated in comparison with uncooked samples. Only 4 cca-miRNAs were found after in vitro digestion. By using scanning electron microscopy (SEM), we also evaluated the extracellular vesicles (EVs) in homogenized artichoke as possible miRNA transporters. However, approximately 81% were degraded after cooking, while the remaining EVs had changed shape from round to elliptical. Finally, we detected no cell-free cca-miRNAs, miRNAs bound to protein complex, and no cca-miRNAs encapsulated in EVs inside Caco-2 cells or in basolateral medium after bioavailability experiments. In conclusion, the data from the present study agrees with recent findings that the human small intestine does not uptake dietary miRNAs from raw or cooked artichoke heads.

Fermented Apulian table olives: Effect of selected microbial starters on polyphenols composition, antioxidant activities and bioaccessibility

The effects of fermentation by autochthonous microbial starters on phenolics composition of Apulian table olives, Bella di Cerignola (BDC), Termite di Bitetto (TDB) and Cellina di Nardò (CEL) were studied, highlighting also the cultivars influence. In BDC with starter, polyphenols amount doubled compared with commercial sample, while in TDB and CEL, phenolics remain almost unchanged. The main phenolics were hydroxytyrosol, tyrosol, verbascoside and luteolin, followed by hydroxytyrosol-acetate detected in BDC and cyanidine-3-glucoside and quercetin in CEL. Scavenger capacity in both DPPH and CAA assays, assessed the highest antioxidant effect for CEL with starters (21.7u202fmg Trolox eq/g FW; 8.5u202fμmol hydroxytyrosol eq/100u202fgu202fFW). The polyphenols were highly in vitro bioaccessible (>60%), although modifications in their profile, probably for combined effect of environment and microorganisms, were noted. Finally, fermented table olives are excellent source of health promoting compounds, since hydroxytyrosol and tyrosol are almost 8 times more than in olive oil.