Mattia Di Nunzio

Pro- and anti-oxidant effects of polyunsaturated fatty acid supplementation in HepG2 cells

PUFA are bioactive nutrients thought to be effective in the prevention of many chronic diseases. PUFA susceptibility to free radical oxidation represents the other side of the coin, and the role of PUFA as pro- or anti-oxidants is still an unanswered question. In this study we supplemented HepG2 cells with different PUFA, and observed different effects on cytotoxicity, oxidation and modulation of antioxidant defenses. These were not simply related to the length of carbon chain, or to the number and position of double bonds. ARA supply evidenced the induction of oxidative damage, while DHA supplemented cells appeared richer in antioxidant defenses. To our knowledge, our study is the first evidencing the different pro- or anti-oxidant effect of different fatty acids when supplemented to cells. Overall, this points out the importance of not generalizing dietary recommendations considering PUFA as one category, but to extend them to the individual fatty acids.

Sugar cane and sugar beet molasses, antioxidant-rich alternatives to refined sugar.

Molasses, the main byproduct of sugar production, is a well-known source of antioxidants. In this study sugar cane molasses (SCM) and sugar beet molasses (SBM) were investigated for their phenolic profile and in vitro antioxidant capacity and for their protective effect in human HepG2 cells submitted to oxidative stress. According to its higher phenolic concentration and antioxidant capacity in vitro, SCM exhibited an effective protection in cells, comparable to or even greater than that of α-tocopherol. Data herein reported emphasize the potential health effects of molasses and the possibility of using byproducts for their antioxidant activity. This is particularly important for consumers in developing countries, as it highlights the importance of consuming a low-price, yet very nutritious, commodity.

N − 3 PUFAs modulate global gene expression profile in cultured rat cardiomyocytes. Implications in cardiac hypertrophy and heart failure

In cardiac cells the effects of n − 3 PUFAs on the whole genome are still unknown despite their recognized cardioprotective effects and ability to modulate gene expression. We have evaluated the effects of n − 3 PUFAs supplementation on the global gene expression profile in cultured neonatal rat cardiomyocytes, detecting many genes related to lipid transport and metabolism among the upregulated ones. Many of the downregulated genes appeared related to inflammation, cell growth, extracellular and cardiac matrix remodelling, calcium movements and ROS generation. Our data allow to speculate that the cardioprotective effect of n − 3 PUFAs is related to a direct modulation of genes in cardiac cells.

Dietary Selenium for the counteraction of oxidative damage: fortified foods or supplements?

Since any significant modification in the Se status, leading to changes in the activity of the seleno-enzymes, may have important consequences on the susceptibility of tissues to oxidative stress, considerable efforts have been made upon increasing Se dietary intake. In this respect, an important debate is still open about the bioavailability and the effectiveness of Se, and more generally nutrients, in supplements compared with foods. Using male Wistar rats, we have compared the effectiveness of two different diets in which an adequate Se content (0.1 mg/kg) was achieved by adding the element as sodium selenite or as component of a lyophilized Se-enriched food, in the counteraction of an oxidative stress induced by intraperitoneal administration of adriamycin. Both Se-enriched diets were able to reduce the consequences of the oxidative stress in liver, mainly by increasing glutathione peroxidase activity. This increase was more evident in rats fed on the diet enriched with the lyophilized food, probably due to the different chemical forms of Se, or to other components of the food itself. Although further studies are needed, data herein presented may contribute to the characterization of the effectiveness of Se from different sources, foods or supplements, in the light of dietary advice to the population concerning improvement of Se intake.

NMR comparison of in vitro digestion of Parmigiano Reggiano cheese aged 15 and 30 months.

The content of essential amino acids is an important aspect for determining the nutritional value of food proteins, but their digestibility is also a key property, deeply affected by food processing. The production of Parmigiano Reggiano cheese is closely related to the nutritional quality of the final product; in particular the high digestibility of its proteins is claimed to be proportional to cheese aging. Two different kinds of Parmigiano Reggiano, young (aged 15 months) and old (aged 30 months), were separately digested using an in vitro system that simulates digestive processes in the mouth, stomach and small intestine. Samples were collected at different stages of digestion and the process of protein hydrolysis was explored and compared by low‐field (LF) and high‐resolution (HR) NMR, together with other biochemical methods. HR‐NMR allowed to simultaneously observe the quantity of free amino acids, peptides and proteins, also giving for these latter qualitative information about their dimension. LF‐NMR, instead, gave the possibility to observe digestion with no treatments whatsoever, thus representing a technique suitable for on‐line measurements. The results pointed out that cheeses with different aging times, although starting from distinct initial compositions, conclude digestion in a similar way, in terms of free amino acids and small organic compounds, but evolve with different kinetics of hydrolysis and peptide formation, discriminating the young from the old cheese. Copyright

Green tea extract selectively activates peroxisome proliferator-activated receptor β/δ in cultured cardiomyocytes

Hypoxia/reoxygenation is one of the causes of the increased expression of inducible NO synthase in cardiomyocytes. In a recent study we demonstrated that a single, high dose of green tea extract (GT) supplemented to the medium of cultured cardiomyocytes just before hypoxia/reoxygenation is able to prevent the increased expression of inducible NO synthase, therefore reducing NO overproduction. In the present study we investigated the mechanism by which GT reduces NO production. Since a molecular mechanism for polyphenol activity has been postulated, and PPAR activation is related to the transcription of the inducible NO synthase gene, we evaluated the activation of PPAR by GT. A moderate GT concentration, supplemented to the cardiomyocyte medium since the initial seeding, selectively activated the PPAR-beta/delta isoform. Furthermore, we observed a reduction in NO production and an increase in total antioxidant activity, indicating that GT components may act on both reactive oxygen species, via an antioxidant mechanism, and NO overproduction. PPAR-beta/delta activation could represent the key event in the reduction of NO production by GT. Although PPAR activation by GT was lower than activation by fenofibrate, it is very interesting to note that it was selective for the beta/delta isoform, at least in neonatal cardiomyocytes.

The foodomics approach for the evaluation of protein bioaccessibility in processed meat upon in vitro digestion

The present work describes a foodomics protocol coupling an in vitro static simulation of digestion to a combination of omics techniques, to grant an overview of the protein digestibility of a meat‐based food, namely Bresaola. The proteolytic activity mediated by the digestive enzymes is evaluated through Bradford and SDS‐PAGE assays, combined to NMR relaxometry and spectroscopy, to obtain information ranging from the microscopic to the molecular level, respectively. The simple proteomics tool adopted here points out that a clear increase of bioaccessible proteins occurs in the gastric phase, rapidly disappearing during the following duodenal digestion. However, SDS‐PAGE and the Bradford assay cannot follow the fate of the digested proteins when the products are sized <5 kDa. Conversely, NMR spectroscopy is able to capture the overall molecular profile of small fragments and peptides, which are mainly formed during the duodenal phase, thus giving the kinetics of the whole digestion process. Time domain NMR relaxometry, finally, detects the swelling phenomenon occurring during the gastric phase, when the digestion fluid enters the meat matrix.

Counteraction of oxidative damage by pomegranate juice: influence of the cultivar

BACKGROUND Pomegranate (Punica granatum) has gained widespread popularity as a potential functional food due to its high phenolics content. Although in different studies pomegranate juice has been shown to exert anti-atherogenic, anti-inflammatory and antioxidant effects, the cultivar-related differences in phenolics content and antioxidant activity must be carefully taken into account when evaluating the health effects. The first aim of this study was to rank the juices of 15 different varieties of pomegranate according to their total phenolic content and antioxidant capacity. Then three juices were selected, better characterised and added to HepG2 cells as a supplement to evaluate the protective effect against induced oxidative stress. RESULTS The cultivars Wonderful, Hicaz and G2 were chosen according to the ranking and the corresponding juices used at two different concentrations as a supplement to HepG2 cells. Overall, all juices were able to protect cells from oxidative damage, but differences that could be related to the different phenolics content and pattern were detected among the three juices. CONCLUSION Our results show the advantage of screening cultivars prior to efficacy studies. This approach can be useful for food companies that focus on the development of food that has added nutritional and health value.

Phytosterol supplementation reduces metabolic activity and slows cell growth in cultured rat cardiomyocytes.

Besides being cholesterol-lowering agents, phytosterols (PS) can inhibit the growth and development of tumours. The anti-neoplastic activity is accounted for by PS incorporation into cell membranes, resulting in the interference of membrane functionality. The similarity between the PS cholesterol-lowering and anti-neoplastic effective doses deserves attention on the possible adverse effects even in non-neoplastic cells. To date, few studies have addressed the clarification of this important issue. In the present study, we supplemented primary, non-neoplastic neonatal rat cardiomyocytes with two different PS concentrations (3 or 6 μg/ml), both within the range of human plasma concentration. Cardiac cells were chosen as an experimental model since the heart has been reported as the target organ for subchronic toxicity of PS. Following supplementation, a dose-dependent incorporation of PS and a decrease in cholesterol content were clearly evidenced. PS did not induce apoptosis but caused a reduction in metabolic activity (measured as 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion) and a slowing down of cell growth. The lower MTT conversion and the similar lactate dehydrogenase release could suggest that PS more efficiently target mitochondria than plasma membrane integrity. The replacement of cholesterol by PS could also have caused the observed slowing down of cell growth and the reduction in metabolic activity, which could rely on the PS increase, cholesterol decrease, or both. The present study is the first report on the effect of PS in cardiac cells, and although it is difficult to translate the obtained results to the health of heart tissue, it raises concerns about the safety of long-term exposure to physiologically relevant PS concentrations.

PUFA and oxidative stress. Differential modulation of the cell response by DHA

Abstract Although an increased dietary intake of long-chain n-3 PUFA is considered an effective preventive strategy, a theoretical concern related to the possible increase of lipid peroxidation induced by a PUFA-rich diet still remains a problem. In this study, the effects of different PUFA (linoleic, α-linolenic, arachidonic, eicosapentaenoic and docosahexaenoic acid) on cytotoxicity, lipid oxidation, and modulation of antioxidant defenses were evaluated in HepG2 cells submitted to an oxidative stress (H2O2). Results clearly evidenced that all supplemented PUFA, but DHA, enhanced cell susceptibility to H2O2. Overall, our results underline that PUFA cannot be considered as a single category but as individual compounds, and research on mechanisms of action and preventive effects should deal with the individual fatty acids, particularly in the case of DHA.