Piero Sestili

Pomegranate peel and fruit extracts: a review of potential anti-inflammatory and anti-infective effects.

ETHNOPHARMACOLOGICAL RELEVANCE Punica granatum L. (Punicaceae) has been used for centuries in many cultures for the prevention and treatment of a wide number of health disorders such as inflammation, diabetes, diarrhea, dysentery, dental plaque and to combat intestinal infections and malarial parasites. AIM OF THE REVIEW This review aims at providing an up-to-date overview of the chemical constituents, traditional uses, phytochemistry, pharmacology and toxicology of Punica granatum L. Moreover, the focus of this review is the possible exploitation of this species to treat different diseases and to suggest future investigations. MATERIALS AND METHODS An extensive and systematic review of the extant literature was carried out, and the data under various sections were identified by using a computerized bibliographic search via PubMed, Web of Science and Google Scholar. All abstracts and full-text articles were examined. The most relevant articles were selected for screening and inclusion in this review. KEY FINDINGS A variety of pomegranate ethnomedical uses have been recorded. Additionally, over the last decade, there has been a dramatic increase of interest in pomegranate as a medicinal and nutritional product due to its n1ewly identified potential health effects, which include treatment and prevention of cancer and cardiovascular diseases. From the toxicological perspective, pomegranate fruit juice, extracts and preparations have been proven to be safe. CONCLUSIONS The ethnopharmacological relevance of pomegranate is fully justified by the most recent findings indicating the fruit is a medicinal and nutritional agent useful for treating a wide range of human disorders and maladies. Further investigations are needed to fully understand the mode of action of the active constituents and to fully exploit pomegranates preventive and therapeutic potential.

Quercetin Prevents DNA Single Strand Breakage and Cytotoxicity Caused By tert-Butylhydroperoxide: Free Radical Scavenging Versus Iron Chelating Mechanism

Although the antioxidant properties of flavonoids are well documented, it is still unclear whether these effects are dependent on radical scavenging or iron chelating activities. By using an experimental approach based on the notion that iron chelators suppress DNA strand scission and cytotoxicity caused by tert-butylhydroperoxide, whereas radical scavenging antioxidants prevent only the latter response, we provide experimental evidence indicating that the most prominent activity of the flavonoid quercetin resides in its ability to chelate iron. This experimental approach can be utilized for the assessment of iron chelation in the biological activity of flavonoids or other antioxidants.

Damage to nuclear DNA induced by Shiga toxin 1 and ricin in human endothelial cells

Ribosome‐inactivating proteins (RIPs) remove a specific adenine from 28S rRNA leading to inactivation of ribosomes and arrest of translation. Great interest as to a possible second physiological substrate for RIPs came from the observation that in vitro RIPs remove adenine from DNA. This paper addresses the problem of nuclear lesions induced by RIPs in human endothelial cells susceptible to the bacterial RIP Shiga toxin 1 and the plant RIP ricin. With both toxins, nuclear DNA damage as evaluated by two independent techniques (alkaline‐halo assay and alkaline filter elution) appears early, concomitant with (ricin) or after (Shiga toxin 1) the inhibition of protein synthesis. At this time, the annexin V binding assay, caspase 3 activity, the formation of typical ≤ 50 Kb DNA fragments, and changes in morphology associated with apoptosis were negative. Furthermore, a block of translation comparable to that induced by RIPs, but obtained with cycloheximide, did not induce nuclear damage. Such damage is consistent with the enzymatic activity (removal of adenine) of RIPs acting in vitro on RNA‐free chromatin and DNA. The results unequivocally indicate that RIPs can damage nuclear DNA in whole cells by means that are not secondary to ribosome inactivation or apoptosis.—Brigotti, M., Alfieri, R., Sestili, P., Bonelli, M., Petronini, P. G., Guidarelli, A., Barbieri, L., Stirpe, F., Sperti, S. Damage to nuclear DNA induced by Shiga toxin 1 and ricin in human endothelial cells. FASEB J. 16, 365–372 (2002)

Pomegranate peel and peel extracts: Chemistry and food features

The present review focuses on the nutritional, functional and anti-infective properties of pomegranate (Punica granatum L.) peel (PoP) and peel extract (PoPx) and on their applications as food additives, functional food ingredients or biologically active components in nutraceutical preparations. Due to their well-known ethnomedical relevance and chemical features, the biomolecules available in PoP and PoPx have been proposed, for instance, as substitutes of synthetic food additives, as nutraceuticals and chemopreventive agents. However, because of their astringency and anti-nutritional properties, PoP and PoPx are not yet considered as ingredients of choice in food systems. Indeed, considering the prospects related to both their health promoting activity and chemical features, the nutritional and nutraceutical potential of PoP and PoPx seems to be still underestimated. The present review meticulously covers the wide range of actual and possible applications (food preservatives, stabilizers, supplements, prebiotics and quality enhancers) of PoP and PoPx components in various food products. Given the overall properties of PoP and PoPx, further investigations in toxicological and sensory aspects of PoP and PoPx should be encouraged to fully exploit the health promoting and technical/economic potential of these waste materials as food supplements.

Creatine as an antioxidant

Creatine monohydrate (Cr), the most diffuse supplement in the sports industry, is receiving greater attention because of its beneficial effects in a wide number of human degenerative diseases and conditions. These effects can be barely explained on the basis of the sole ergogenic role of the Cr/CrP system. Indeed, a wide number of research articles indicate that Cr is capable of exerting multiple, non-energy related, effects on diverse and relevant cellular targets. Among these effects, the antioxidant activity of Cr emerges as an additional mechanism which is likely to play a supportive role in the Cr-cytoprotection paradigm.

Apoptosis and necrosis following exposure of U937 cells to increasing concentrations of hydrogen peroxide: the effect of the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide.

A 3-hr exposure of U937 cells to hydrogen peroxide (H2O2) followed by a 6-hr posttreatment incubation in fresh culture medium promotes apoptosis or necrosis, depending on the oxidant concentration. Addition of 3-aminobenzamide (3AB) during the recovery phase prevented necrosis and caused apoptosis. 3AB did not, however, affect the apoptotic response of cells treated with apogenic concentrations of H2O2. Cells exposed for 3 hr to 1.5 mM H2O2, while showing some signs of suffering, maintained a normal nuclear organization and good organelle morphology. At the biochemical level, the oxidant promoted the formation of Mb-sized DNA fragments and rapidly depleted both the adenine nucleotide and non-protein sulphydryl pools, which did not recover during posttreatment incubation in the absence or presence of 3AB. These results allow a novel interpretation of the concentration-dependent switch from apoptosis to necrosis. We propose that H2O2 activates the apoptotic response at the early times of peroxide exposure and that this process can be completed, or inhibited, during the posttreatment incubation phase. Inhibition of apoptosis leads to necrosis and can be prevented by 3AB via a mechanism independent of inhibition of poly(ADP-ribose)polymerase. As a corollary, the necrotic response promoted by high concentrations of H2O2 in U937 cells appears to be the result of specific inhibition of the late steps of apoptosis.

Inhibition of AMPK signalling by doxorubicin: at the crossroads of the cardiac responses to energetic, oxidative, and genotoxic stress

AIMS Cardiotoxic side effects of anthracyclines, the most widely used anticancer drugs, are well documented, while mechanisms involved are not fully elucidated. The cellular energy sensor and regulator AMP-activated protein kinase (AMPK) was suggested as a putative mediator of cardiotoxicity of doxorubicin, the leading anthracycline drug, by our earlier work. Here, we study the interference of doxorubicin with AMPK signalling and potentially involved mechanisms. METHODS AND RESULTS Effects of doxorubicin on cell signalling are studied in isolated Langendorff-perfused Wistar rat hearts and in hearts from doxorubicin-treated Wistar rats. In both models, doxorubicin induces energetic, oxidative, and genotoxic stress. Despite energy depletion and unaffected AMPK upstream signalling, doxorubicin does not activate the AMPK pathway and even reduces basal phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase. In contrast, oxidative and genotoxic stress do activate pro-survival mitogen-activated protein kinase (MAPK) and Akt pathways, the latter via DNA-dependent protein kinase activation triggered by DNA damage. Combined inhibition of AMPK and activation of Akt and MAPK lead to activation of growth-stimulating mammalian target of rapamycin (mTOR) signalling. CONCLUSION Our results suggest that in the doxorubicin-challenged heart, a combined energetic, oxidative, and genotoxic stress elicits a specific, hierarchical response where AMPK is inhibited at least partially by the known negative cross-talk with Akt and MAPK pathways, largely triggered by DNA damage signalling. Although such signalling can be protective, e.g. by limiting apoptosis, it primarily induces a negative feedback that increases cellular energy deficits, and via activation of mTOR signalling, it also contributes to the pathological cardiac phenotype in chronic doxorubicin toxicity.

Creatine supplementation prevents the inhibition of myogenic differentiation in oxidatively injured C2C12 murine myoblasts.

Creatine (Cr), one of the most popular nutritional supplements among athletes, has been recently shown to prevent the cytotoxicity caused by different oxidative stressors in various mammalian cell lines, including C2C12 myoblasts, via a direct antioxidant activity. Here, the effect of Cr on the differentiating capacity of C2C12 cells exposed to H(2)O(2) has been investigated. Differentiation into myotubes was monitored using morphological, ultrastructural, and molecular techniques. Treatment with H(2)O(2) (1 h) not only caused a significant (30%) loss of cell viability, but also abrogated the myogenic ability of surviving C2C12. Cr-supplementation (24 h prior to H(2)O(2) treatment) was found to prevent these effects. Interestingly, H(2)O(2)-challenged cells preconditioned with the established antioxidants trolox or N-acetyl-cysteine, although cytoprotected, did not display the same differentiating ability characterizing oxidatively-injured, Cr-supplemented cells. Besides acting as an antioxidant, Cr increased the level of muscle regulatory factors and IGF1 (an effect partly refractory to oxidative stress), the cellular availability of phosphocreatine and seemed to exert some mitochondrially-targeted protective activity. It is concluded that Cr preserves the myogenic ability of oxidatively injured C2C12 via a pleiotropic mechanism involving not only its antioxidant capacity, but also the contribution to cell energy charge and effects at the transcriptional level which common bona fide antioxidants lack.

Prevention of necrosis and activation of apoptosis in oxidatively injured human myeloid leukemia U937 cells

A 3 h exposure to 1 mM H2O2 followed by 6 h post‐challenge growth in peroxide‐free medium induces necrosis in U937 cells. Addition of the poly(ADP‐ribose)polymerase inhibitor 3‐aminobenzamide during recovery prevents necrosis and triggers apoptosis, as shown by the appearance of apoptotic bodies, extensive blebbing and formation of multimeric DNA fragments as well as 50 kb double stranded DNA fragments. Thus, the same initial damage can be a triggering event for both apoptotic and necrotic cell death. Furthermore, necrosis does not appear to be a passive response to overwhelming damage.

Creatine as a compatible osmolyte in muscle cells exposed to hypertonic stress.

Exposure of C2C12 muscle cells to hypertonic stress induced an increase in cell content of creatine transporter mRNA and of creatine transport activity, which peaked after about 24 h incubation at 0.45 osmol (kg H2O)−1. This induction of transport activity was prevented by addition of either cycloheximide, to inhibit protein synthesis, or of actinomycin D, to inhibit RNA synthesis. Creatine uptake by these cells is largely Na+ dependent and kinetic analysis revealed that its increase under hypertonic conditions resulted from an increase in Vmax of the Na+‐dependent component, with no significant change in the Km value of about 75 μmol l−1. Quantitative real‐time PCR revealed a more than threefold increase in the expression of creatine transporter mRNA in cells exposed to hypertonicity. Creatine supplementation significantly enhanced survival of C2C12 cells incubated under hypertonic conditions and its effect was similar to that obtained with the well known compatible osmolytes, betaine, taurine and myo‐inositol. This effect seemed not to be linked to the energy status of the C2C12 cells because hypertonic incubation caused a decrease in their ATP content, with or without the addition of creatine at 20 mmol l−1 to the medium. This induction of creatine transport activity by hypertonicity is not confined to muscle cells: a similar induction was shown in porcine endothelial cells.