PROTECTIVE ROLE OF DIETARY BIOACTIVE COMPOUNDS: MECHANISMS AND HYPOTHESIS

Scientific evidences support the protective role of diets rich in fruit and vegetables against chronic diseases, like cancer and cardiovascular disease. In particular two classes of chemopreventive phytochemicals, i.e. isothiocyanates presence in Cruciferae and anthocyanins (ACNs) presence in berries and other colored fruits and vegetables, are considered in this thesis. Cruciferae and especially Brassica genus contain high concentration of constituents with antioxidant properties (e.g. carotenoids, vitamin C, folate) as well as glucosinolate precursors of isothiocyanates (ITCs) and indoles that modulate xenobiotic biotransformation enzymes, such as Glutathione S-Transferase (GST). The protective effect of broccoli (Brassica oleracea L. var. italica) was tested through two different study protocols: a regular-intake intervention study with daily consumption of one serving of steamed broccoli for 10 days and a single-meal study (i.e. single consumption of broccoli, 250g). Analyzed broccoli resulted to be a good source of antioxidant compounds (carotenoids, vitamin C) and glucosinolates as ITCs. Subjects involved in the study were young male smokers recruited on the basis of anthropometric characteristics, food habits and their GSTM1 genotype. Thirty subjects were enrolled for the regular-intake study. A single blind randomized cross-over experimental design was scheduled. Fasting blood samples were collected at the beginning and at the end of each treatment period (0, 10, 30, 40 days). Concentration of carotenoids, lutein, β-carotene and folate was assessed in plasma and serum samples. Lymphocytes were used for the determination of biomarkers of oxidative stress: cell resistance against oxidative stress, endogenous DNA damage (i.e. oxidized purines), in vitro DNA repair activity and mRNA expression of OGG1, NUDT1 and HO-1 levels. Cell protection against H2O2-induced DNA damage was higher after broccoli diet with respect to control diet in the whole group of subjects. Folate and lutein concentrations increased significantly after broccoli diet. Broccoli intake caused a more consistent and significant effect of protection against DNA damage in GSTM1 null (-27.6%) subjects compared to GSTM1 positive (-13.1%) individuals (p<0.05). Oxidized purines decreased significantly (p<0.05) after broccoli intake (-22.6%) while no effect of polymorphism was observed. DNA repair activity and OGG1, HO-1 and NUDT1 mRNA expression levels did not differ throughout the intervention study. Twelve volunteers were selected for the single-meal study. Fasting blood samples were collected before broccoli consumption and after 3, 6, 8 and 24 h from broccoli intake in order to evaluate: cell resistance to oxidative DNA damage, bioavailability of bioactive compounds (folate, lutein, β-carotene and vitamin C) and total GST activity. In particular, serum folate, plasma vitamin C and β-carotene concentrations significantly increased. Furthermore, plasma GST activity increased significantly in individuals with GSTM1 positive genotype at 6 h with respect to 3 h and 24 h but not compared to baseline. Interestingly, ex-vivo induced DNA damage was significantly reduced after 24 h from broccoli consumption. In conclusion, these two studies demonstrated that 10 days of consumption of steamed broccoli could improve defence against DNA damage, without affecting repair activity in young healthy smokers. Moreover, a single portion of steamed broccoli was able to decrease DNA damage and to modulate GST activity. Even if preliminary, our data suggest a “diet/genetic” interaction. Delphinidin-3-glucoside (Dp-3-glc) and cyanidin-3-glucoside (Cy-3-glc) are two ACNs able to inhibit tumor cell proliferation. An in vitro study was designed to investigate the effect of Dp-3-glc and Cy-3-glc on the angiogenic and Dp-3-glc on the procoagulant activities of human microvascular endothelial cells (HMEC-1). HMEC-1 were incubated for up to 24h with culture media ± ACNs (0.1, 1, 10, 100 µM) alone or in combination with purified proangiogenic factor (VEGF), or bacterial endotoxin (LPS). Angiogenesis was evaluated by the capillary-like tube formation in Matrigel and the wound healing assay; while the pro-coagulant activity was tested by the thrombin generation (TG) assay and Tissue Factor (TF) expression as antigen, one-stage recalcification assay and mRNA quantitation. Results showed that Dp-3-glc was able to inhibit angiogenesis in resting cells and in VEGF-stimulated conditions at 100 µM; interestingly, the inhibition of the migratory VEGF-dependent stimulus started at the Dp-3-glc concentration of 1 µM. In the TG assay Dp-3-glc significantly contrasted with the pro-thrombotic stimulus of LPS starting from 10 µM on HMEC-1 intact monolayer and TF expression at 100 µM concentration. Differently, Cy-3-glc did not show any effect on angiogenesis. In conclusion, this study, in terms of anti-angiogenic and anti-coagulant properties of Dp-3-glc make this compound a potential cancer chemopreventive agent. Together our results, from both in vivo studies on humans and in vitro cell culture models, support potential health benefits derived from the intake of fruit (coloured berries) and vegetable (broccoli).