Cristina Angeloni

Sulforaphane as a Potential Protective Phytochemical against Neurodegenerative Diseases

A wide variety of acute and chronic neurodegenerative diseases, including ischemic/traumatic brain injury, Alzheimers disease, and Parkinsons disease, share common characteristics such as oxidative stress, misfolded proteins, excitotoxicity, inflammation, and neuronal loss. As no drugs are available to prevent the progression of these neurological disorders, intervention strategies using phytochemicals have been proposed as an alternative form of treatment. Among phytochemicals, isothiocyanate sulforaphane, derived from the hydrolysis of the glucosinolate glucoraphanin mainly present in Brassica vegetables, has demonstrated neuroprotective effects in several in vitro and in vivo studies. In particular, evidence suggests that sulforaphane beneficial effects could be mainly ascribed to its peculiar ability to activate the Nrf2/ARE pathway. Therefore, sulforaphane appears to be a promising compound with neuroprotective properties that may play an important role in preventing neurodegeneration.

Neuroprotective effects of anthocyanins and their in vivo metabolites in SH-SY5Y cells

Recent in vivo studies have highlighted an important role for the neuroprotective actions of dietary anthocyanins. However, one consistent result of these studies is that the systemic bioavailability of anthocyanins, including cyanidin 3-O-glucopyranoside (Cy-3G), is very poor. Cy-3G has been demonstrated to be highly instable at physiological pH, so its in vivo metabolites, such as the aglycon cyanidin (Cy) and protocatechuic acid (PA), may be responsible for both the antioxidant activitiy and the neuroprotective effects observed in vivo. Therefore, we investigated the protective effects of Cy-3G, Cy and PA against H(2)O(2)-induced oxidative stress in a human neuronal cell line (SH-SY5Y). We determined their ability to counteract reactive oxygen species (ROS) formation and to inhibit apoptosis in terms of mitochondrial functioning loss and DNA fragmentation induced by H(2)O(2). We demonstrated that pretreatment of SH-SY5Y cells with Cy-3G, Cy and PA inhibits H(2)O(2)-induced ROS formation at different cellular levels: Cy-3G at membrane level, PA at cytosolic level and Cy at both membrane and cytosolic levels. In addition, Cy showed a higher antioxidant activity at membrane and cytosolic level than Cy-3G and PA, respectively. Interestingly, both Cy and PA, but not Cy-3G, could inhibit H(2)O(2)-induced apoptotic events, such as mitochondrial functioning loss and DNA fragmentation. These results suggest that Cy and PA may be considered as neuroprotective molecules and may play an important role in brain health promotion. These in vitro findings should encourage further research in animal models of neurological diseases to explore the potential neuroprotective effects of compounds generated during in vivo metabolism of anthocyanins.

Modulation of Phase II Enzymes by Sulforaphane: Implications for Its Cardioprotective Potential

Oxidative stress plays a major role in the pathophysiology of cardiac disorders, but the experimental data on the protective effects of exogenous antioxidants are controversial. A promising cardioprotective strategy may be through the induction of the endogenous antioxidants and phase II enzymes by chemical inducers. Sulforaphane is an isothiocyanate derived from cruciferous vegetables, and it has gained attention mainly as a potential chemopreventive agent in part through the induction of detoxifying enzymes. Accordingly, this study was undertaken to investigate the time-dependent induction of gene transcription, protein expression, and enzyme activity of antioxidant and phase II enzymes [glutathione reductase, glutathione-S-transferase, glutathione peroxidase, NAD(P)H:quinone oxidoreductase-1, thioredoxin reductase] by sulforaphane in cultured rat neonatal cardiomyocytes. The potential cardioprotective action of sulforaphane was confirmed by the decrease in intracellular reactive oxygen species production, the increase in cell viability, and the decrease in DNA fragmentation after long-term treatment accompanied by the induction of antioxidants and phase II enzymes in cardiomyocytes.

Inflammation-Induced Alteration of Astrocyte Mitochondrial Dynamics Requires Autophagy for Mitochondrial Network Maintenance

Accumulating evidence suggests that changes in the metabolic signature of astrocytes underlie their response to neuroinflammation, but how proinflammatory stimuli induce these changes is poorly understood. By monitoring astrocytes following acute cortical injury, we identified a differential and region-specific remodeling of their mitochondrial network: while astrocytes within the penumbra of the lesion undergo mitochondrial elongation, those located in the core-the area invaded by proinflammatory cells-experience transient mitochondrial fragmentation. In brain slices, proinflammatory stimuli reproduced localized changes in mitochondrial dynamics, favoring fission over fusion. This effect was triggered by Drp1 phosphorylation and ultimately resulted in reduced respiratory capacity. Furthermore, maintenance of the mitochondrial architecture critically depended on the induction of autophagy. Deletion of Atg7, required for autophagosome formation, prevented the reestablishment of tubular mitochondria, leading to marked reactive oxygen species accumulation and cell death. Thus, our data reveal autophagy to be essential for regenerating astrocyte mitochondrial networks during inflammation.

Green tea protection of hypoxia/reoxygenation injury in cultured cardiac cells

Antioxidant-rich diets exert a protective effect in diseases involving oxidative damage. Among dietary components, green tea is an excellent source of antioxidants. In this study, cultured neonatal rat cardiomyocytes were used to clarify the protective effect of a green tea extract on cell damage and lipid peroxidation induced by different periods of hypoxia followed by reoxigenation. Cultures of neonatal rat cardiomyocytes were exposed to 2--8 hr hypoxia, eventually followed by reoxygenation, in the absence or presence of alpha-tocopherol or green tea. LDH release and the production of conjugated diene lipids were measured, and appeared linearly related to the duration of hypoxia. During hypoxia, both LDH release and conjugated diene production were reduced by alpha-tocopherol and, in a dose dependent manner, by green tea, the 50 &mgr;g/ml being the most effective dose. Reoxygenation caused no further increase in LDH leakage, while it caused a significant increase in conjugate dienes, which absolute value was lower in antioxidant supplemented cells. Anyway, the ratio between conjugated diene production after hypoxia and after reoxygenation was similar in all groups, indicating that the severity of free radical-induced reoxygenation injury is proportional to the severity of previous hypoxic injury. Since hypoxic damage is reduced by alpha-tocopherol and green tea, our data suggest that any nutritional intervention to attenuate reoxygenation injury must be directed toward the attenuation of the hypoxic injury. Therefore, recommendations about a high dietary intake of antioxidants may be useful not only in the prevention, but also in the reduction of cardiac injury following ischemia.

Sulforaphane as an inducer of glutathione prevents oxidative stress-induced cell death in a dopaminergic-like neuroblastoma cell line

The total GSH depletion observed in the substantia nigra (SN) appears to be responsible for subsequent oxidative stress (OS), mitochondrial dysfunction, and dopaminergic cell loss in patients with Parkinson’s disease. A strategy to prevent the OS of dopaminergic cells in the SN may be the use of chemopreventive agents as inducers of endogenous GSH, antioxidant and phase 2 enzymes. In this study, we demonstrated that treatment of the dopaminergic‐like neuroblastoma SH‐SY5Y cell line with sulforaphane (SF), a cruciferous vegetables inducer, resulted in significant increases of total GSH level, NAD(P)H : quinone oxidoreductase‐1, GSH‐transferase and ‐reductase, but not GSH‐peroxidase, catalase and superoxide dismutase activities. Further, the elevation of GSH levels, GSH‐transferase and NAD(P)H:quinone oxidoreductase‐1 activities was correlated to an increase of the resistance of SH‐SY5Y cells to toxicity induced by H2O2 or 6‐hydroxydopamine (6‐OHDA). The pre‐treatment of SH‐SY5Y cells with SF was also shown to prevent various apoptotic events (mitochondrial depolarization, caspase 9 and 3 activation and DNA fragmentation) and necrosis elicited by 6‐OHDA. Further, the impairment of antioxidant capacity and reactive oxygen species formation at intracellular level after exposure to 6‐OHDA was effectively counteracted by pre‐treatment with SF. Last, both the cytoprotective and antioxidant effects of SF were abolished by the addition of buthionine sulfoximine supporting the main role of GSH in the neuroprotective effects displayed by SF. These findings show that SF may play a role in preventing Parkinson’s disease.

Relevance of apple consumption for protection against oxidative damage induced by hydrogen peroxide in human lymphocytes.

In a single-dosing crossover study, we investigated the ability of apple fruit consumption to protect human lymphocytes against peroxide-induced damage to DNA. Six healthy, non-smoking male volunteers were placed for 2d on an antioxidant-poor (AP) diet. After 48h of AP diet, the volunteers were required to consume a homogenate obtained from 600g of red delicious unpeeled apples or water (500 ml); blood samples were collected 0, 3, 6 and 24 h post-consumption. To evaluate whether the apple intake was sufficient to restore resistance of DNA to oxidative damage, for each subject at any time point the plasma total antioxidant activity, reactive oxygen species (ROS) formation and induction of micronuclei (MN) in isolated lymphocytes following hydrogen peroxide (H2O2) treatment were measured. Results indicated a significant inhibition (58%, P <0.05) of H2O2-induced MN frequency in the plasma samples collected at 3 h after apple consumption, as compared with plasma samples collected at 0 h (4.17 (SD 1.83) v. 9.85 (SD 1.87) MN/1000 binucleated (BN) cells, respectively). A gradual return towards the value observed at 0 h was recorded starting from 6 to 24 h. MN frequency induced by H2O2 was significantly influenced by plasma total antioxidant activity (r = -0.95, P <0.05) and by the increase of intracellular ROS formation (r = 0.88, P <0.05). These findings suggest that the consumption of whole apple provides a useful dietary source of active scavengers to protect cells and tissue from oxidative stress and related DNA injury.

Doxorubicin induces early lipid peroxidation associated with changes in glucose transport in cultured cardiomyocytes

Doxorubicin (DOX) has not only chronic, but also acute toxic effects in the heart, ascribed to the generation of reactive oxygen species (ROS). Focusing on the DOX-induced early biochemical changes in rat cardiomyocytes, we demonstrated that lipid peroxidation is an early event, in fact conjugated diene production increased after 1-h DOX exposure, while cell damage, evaluated as lactate dehydrogenase (LDH) release, was observed only later, when at least one third of the cell antioxidant defences were consumed. Cell pre-treatment with alpha-tocopherol (TC) inhibited both conjugated diene production and LDH release. In cardiomyocytes, DOX treatment caused a maximal increase in glucose uptake at 1 h, demonstrating that glucose transport may represent an early target for DOX. At longer times, as the cell damage become significant, the glucose uptake stimulation diminished. Immunoblotting of glucose transporter isoform GLUT1 in membranes after 1-h DOX exposure revealed an increase in GLUT1 amount similar to the increase in transport activity; both effects were inhibited by alpha TC. Early lipid peroxidation evokes an adaptive response resulting in an increased glucose uptake, presumably to restore cellular energy. The regulation of nutrient transport mechanisms in cardiomyocytes may be considered an early event in the development of the cardiotoxic effects of the anthracycline.

Sulforaphane protects cortical neurons against 5-S-cysteinyl-dopamine-induced toxicity through the activation of ERK1/2, Nrf-2 and the upregulation of detoxification enzymes.

The degeneration of dopaminergic neurons in the substantia nigra has been linked to the formation of the endogenous neurotoxin 5-S-cysteinyl-dopamine. Sulforaphane (SFN), an isothiocyanate derived from the corresponding precursor glucosinolate found in cruciferous vegetables has been observed to exert a range of biological activities in various cell populations. In this study, we show that SFN protects primary cortical neurons against 5-S-cysteinyl-dopamine induced neuronal injury. Pre-treatment of cortical neurons with SFN (0.01-1 microM) resulted in protection against 5-S-cysteinyl-dopamine-induced neurotoxicity, which peaked at 100 nM. This protection was observed to be mediated by the ability of SFN to modulate the extracellular signal-regulated kinase 1 and 2 and the activation of Kelch-like ECH-associated protein 1/NF-E2-related factor-2 leading to the increased expression and activity of glutathione-S-transferase (M1, M3 and M5), glutathione reductase, thioredoxin reductase and NAD(P)H oxidoreductase 1. These data suggest that SFN stimulates the NF-E2-related factor-2 pathway of antioxidant gene expression in neurons and may protect against neuronal injury relevant to the aetiology of Parkinsons disease.

H2O2 preconditioning modulates phase II enzymes through p38 MAPK and PI3K/Akt activation.

Ischemic preconditioning is a complex cardioprotective phenomenon that involves adaptive changes in cells and molecules and occurs in a biphasic pattern: an early phase after 1-2 h and a late phase after 12-24 h. While it is widely accepted that reactive oxygen species are strongly involved in triggering ischemic preconditiong, it is not clear if they play a major role in the early or late phase of preconditioning and which are the mechanisms involved. The present study was designed to investigate the mechanisms behind H(2)O(2)-induced cardioprotection in rat neonatal cardiomyocytes. We focused on antioxidant and phase II enzymes and their modulation by protein kinase signaling pathways and nuclear-factor-E(2)-related factor-1 (Nrf1) and Nrf2. H(2)O(2) preconditioning was able to counteract oxidative stress more effectively in the late than in the early phase of adaptation. In particular, H(2)O(2) preconditioning counteracted oxidative stress-induced apoptosis by decreasing caspase-3 activity, increasing Bcl2 expression and selectively increasing the expression and activity of antioxidant and phase II enzymes through Nrf1 and Nrf2 translocation to the nucleus. The downregulation of Nrf1 and Nrf2 by small interfering RNA reduced the expression level of phase II enzymes. Specific inhibitors of phosphatidylinositol 3-kinase/Akt and p38 MAPK activation partially reduced the cardioprotection elicited by H(2)O(2) preconditioning and the induction and activity of phase II enzymes. These findings demonstrate, for the first time, a key role for Nrf1, and not only for Nrf2, in the induction of phase II enzymes triggered by H(2)O(2) preconditioning.