Eleonora Pagnotta

Dose-dependent effects of R-sulforaphane isothiocyanate on the biology of human mesenchymal stem cells, at dietary amounts, it promotes cell proliferation and reduces senescence and apoptosis, while at anti-cancer drug doses, it has a cytotoxic effect

Brassica vegetables are attracting a great deal of attention as healthy foods because of the fact that they contain substantial amounts of secondary metabolite glucosinolates that are converted into isothiocyanates, such as sulforaphane [(−)1-isothiocyanato-4R-(methylsulfinyl)-butane] (R-SFN), through the actions of chopping or chewing the vegetables. Several studies have analyzed the biological and molecular mechanisms of the anti-cancer activity of synthetic R,S-sulforaphane, which is thought to be a result of its antioxidant properties and its ability to inhibit histone deacetylase enzymes (HDAC). Few studies have addressed the possible antioxidant effects of R-SFN, which could protect cells from the free radical damage that strongly contribute to aging. Moreover, little is known about the effect of R-SFN on stem cells whose longevity is implicated in human aging. We evaluated the effects of R-SFN on the biology on human mesenchymal stem cells (MSCs), which, in addition to their ability to differentiate into mesenchymal tissues, support hematopoiesis, and contribute to the homeostatic maintenance of many organs and tissues. Our investigation found evidence that low doses of R-SFN promote MSCs proliferation and protect them from apoptosis and senescence, while higher doses have a cytotoxic effect, leading to the induction of cell cycle arrest, programmed cell death and senescence. The beneficial effects of R-SFN may be ascribed to its antioxidant properties, which were observed when MSC cultures were incubated with low doses of R-SFN. Its cytotoxic effects, which were observed after treating MSCs with high doses of R-SFN, could be attributed to its HDAC inhibitory activity. In summary, we found that R-SFN, like many other dietary supplements, exhibits a hormetic behavior; it is able to induce biologically opposite effects at different doses.

Comparison of bioactive phytochemical content and release of isothiocyanates in selected brassica sprouts

The consumption of brassica sprouts as raw vegetables provides a fair amount of glucosinolates (GLs) and active plant myrosinase, which enables the breakdown of GLs into health-promoting isothiocyanates (ITCs). This study reports the determination of the main constituents related to human health found in edible sprouts of two Brassica oleracea varieties, broccoli and Tuscan black kale, and two Raphanus sativus varieties, Daikon and Sango. Radish sprouts exhibited the highest ability to produce ITCs, with Daikon showing the greatest level of conversion of GLs into bioactive ITCs (96.5%), followed by Sango (90.0%). Tuscan black kale gave a value of 68.5%, whereas broccoli displayed the lowest with 18.7%. ITCs were not the exclusive GL breakdown products in the two B. oleracea varieties, since nitriles were also produced, thus accounting for the lower conversion observed. Measuring the release of plant ITCs is a valuable tool in predicting the potential level of exposure to these bioactive compounds after the consumption of raw brassica sprouts.

Low concentrations of isothiocyanates protect mesenchymal stem cells from oxidative injuries, while high concentrations exacerbate DNA damage

Isothiocyanates (ITCs) are molecules naturally present in many cruciferous vegetables (broccoli, black radish, daikon radish, and cauliflowers). Several studies suggest that cruciferous vegetable consumption may reduce cancer risk and slow the aging process. To investigate the effect of ITCs on cellular DNA damage, we evaluated the effects of two different ITCs [sulforaphane (SFN) and raphasatin (RPS)] on the biology of human mesenchymal stem cells (MSCs), which, in addition to their ability to differentiate into mesenchymal tissues, contribute to the homeostatic maintenance of many organs. The choice of SFN and RPS relies on two considerations: they are among the most popular cruciferous vegetables in the diet of western and eastern countries, respectively, and their bioactive properties may differ since they possess specific molecular moiety. Our investigation evidenced that MSCs incubated with low doses of SFN and RPS show reduced in vitro oxidative stress. Moreover, these cells are protected from oxidative damages induced by hydrogen peroxide, while no protection was evident following treatment with the UV ray of a double strand DNA damaging drug, such as doxorubicin. High concentrations of both ITCs induced cytotoxic effects in MSC cultures and further increased DNA damage induced by peroxides. In summary, our study suggests that ITCs, at low doses, may contribute to slowing the aging process related to oxidative DNA damage. Moreover, in cancer treatment, low doses of ITCs may be used as an adjuvant to reduce chemotherapy-induced oxidative stress, while high doses may synergize with anticancer drugs to promote cell DNA damage.

Effect of sprout extract from Tuscan black cabbage on xenobiotic-metabolizing and antioxidant enzymes in rat liver.

In recent years, health protection by natural products has received considerable attention, and a multitude of nutraceuticals have been characterized and their use promoted. Dietary consumption of Cruciferous vegetables, rich in glucosinolates (GLs), and their myrosinase-mediated hydrolysis products isothiocyanates (ITCs), were associated with reductions in cancer risk. In this study, the chemo-preventive potential of sprout extract of Tuscan black cabbage (Brassica oleracea L. var. acephala subvar. Laciniata L.) (TBCSE), through modulation of the xenobiotic-metabolizing apparatus and antioxidant defenses, was investigated in Sprague-Dawley rat liver. TBCSE was administered either orally or intraperitoneally, at a dose of 15mg/kg b.w., daily for twenty-one consecutive days, in the absence or presence of exogenous myrosinase, β-thioglucoside glucohydrolase (MYR), to distinguish the effects of intact GLs and ITCs, in the context of the extract. A complex, mild modulation pattern of P450-related monooxygenases was observed, mainly regarding CYP content (up to 36% loss), NADPH cytochrome (P450) c-reductase (up to 26% loss), CYP1A1 (up to 23% loss), but no evident distinctions among the effects of the extracts containing GLs or ITCs, were noted. In contrast, significant inductions of phase-II enzymes (up to 107% for UDP-glucuronosyl-transferase, and up to 36% for glutathione S-transferase) were recorded only where the GLs to ITCs conversion had occurred. A boosting effect on catalase (up to 38%), NAD(P)H:quinone reductase (up to 70%), glutathione reductase and glutathione peroxidase (up to 10%) was also recorded, suggesting an indirect antioxidant capacity of the extracts. Overall, the general phase-I inhibition, together with the up-regulation of detoxifying phase-II and antioxidant enzymes, exerted by the TBCSE supplementation, seem to be in line with the classical chemopreventive theory, but whether the addition of exogenous MYR is relevant, still remains to be clarified. These results are in support of the potential health-promoting application of TBCSE, as a nutraceutical.

Biocontrol of Monilinia laxa by Aureobasidium pullulans strains: Insights on competition for nutrients and space

Two Aureobasidium pullulans strains (L1 and L8), able to prevent postharvest fruit decay, were evaluated in order to elucidate how the competition for nutrients and space was involved in their activity against Monilinia laxa, the causal agent of peach brown rot. The competition for nutrients was studied by co-culturing pathogen conidia and antagonists in different conditions of nutrient availability and avoiding contact between them. Both antagonists prevented the germination of conidia of M. laxa in water, reducing germination rate by >35%. However, L1 and L8 showed the lowest inhibition of conidial germination in peach juice at 5%, with a reduction of 12.6% and 13.9% respectively. HPLC amino acid analysis of peach juice revealed that the addition of the yeast suspension greatly modified their composition: asparagine was completely depleted soon after 12h of incubation and was probably hydrolyzed to aspartic acid by the yeasts, as aspartic acid content markedly increased. Pure asparagine and aspartic acid were tested by in vitro trials at the concentrations found in peach juice: both influenced M. laxa growth, but in opposite ways. Asparagine stimulated pathogen growth; conversely, amended medium with aspartic acid significantly inhibited the conidia germination and mycelial development of M. laxa. Scanning Electron Microscopy revealed that both strains showed a great capability to compete with M. laxa for space (starting 8h after treatment), colonizing the wound surface and inhibiting pathogen growth. This study clearly showed that A. pullulans L1 and L8 strains could compete with M. laxa for nutrients and space; this mode of action may play an important role in the antagonistic activity, especially in the first hours of tritrophic host-pathogen-antagonist interaction, although several other mechanisms can interact each other.