Research article
“In vitro” is a Latin phrase meaning “in glass”. In biology and medicine, it describes procedures, experiments, or observations performed outside of a living organism—such as in a test tube, petri dish, or culture flask.
Serena Benedetti , Simona Catalani , Francesco Palma , Franco Canestrari
CELLFOOD® (CF) is an innovative nutritional supplement containing 78 ionic/colloidal trace elements and minerals combined with 34 enzymes and 17 amino acids, all suspended in a solution of deuterium sulfate. The aim of this study was to investigate, for the first time, the antioxidant properties of CF in vitro in different model systems.
Three pathophysiologically relevant oxidants were chosen to evaluate CF protection against oxidative stress: hydrogen peroxide, peroxyl radicals, and hypochlorous acid. Both biomolecules (GSH and plasmid DNA) and circulating cells (erythrocytes and lymphocytes) were used as targets of oxidation.
CF protected, in a dose-dependent manner, both GSH and DNA from oxidation by preserving reduced GSH thiol groups and supercoiled DNA integrity, respectively. At the same time, CF protected erythrocytes from oxidative damage by reducing cell lysis and GSH intracellular depletion after exposure to the oxidant agents. In lymphocytes, CF reduced the intracellular oxidative stress induced by the three oxidants in a dose-dependent manner.
The overall in vitro protection of biomolecules and cells against free radical attacks suggests that CF might be a valuable coadjuvant in the prevention and treatment of various physiological and pathological conditions related to oxidative stress, from aging to atherosclerosis, from neurodegeneration to cancer.
► CELLFOOD® is a novel nutritional supplement based on a solution of deuterium sulfate, enzymes, amino acids, and minerals. ► The efficacy of CF has been evidenced in fibromyalgic subjects and athletes, possibly due to an antioxidant protection. ► In this study we investigate for the first time the antioxidant properties of CF in vitro in different model systems. ► CF protects both biomolecules (GSH and DNA) and cells (erythrocytes and lymphocytes) from oxidation. ► CF may be a valuable coadjuvant in the prevention and treatment of various conditions related to oxidative stress.
AAPH, 2,2′-azobis(2-amidinopropane) hydrochloride; BAP, biological antioxidant potential; CF, CELLFOOD®;
DCFH-DA, 2′,7′-dichlorofluorescin diacetate; DTNB, 5,5-dithio-bis-2-nitrobenzoic; GSH, glutathione; Hb, hemoglobin;
H2O2, hydrogen peroxide; HOCl, hypochlorous acid; mtDNA, mitochondrial DNA; NaOCl, sodium hypochlorite;
PBS, phosphate-buffered saline; RBC, red blood cells; ROS, reactive oxygen species
CELLFOOD®; Nutritional supplement; Oxidative damage; Antioxidant protection; Red blood cells; DNA
Increased oxidative stress generally describes a condition in which cellular antioxidant defenses are inadequate to completely inactivate the free radicals generated by an excessive production of reactive oxygen species (ROS), loss of antioxidant defenses, or both (Halliwell and Gutteridge, 1999). A major consequence of oxidative stress is damage to nucleic acids, lipids, and proteins, which can severely compromise cell health and viability, ultimately leading to cell death and the development of disease (Dalle-Donne et al., 2006).
Among macromolecules, mitochondrial DNA (mtDNA) is highly exposed to ROS produced by electron leakage during oxidative phosphorylation (Yakes and Van, 1997). Moreover, it is thought to be more susceptible to oxidative damage than nuclear DNA (nDNA) due to the lack of histones (Dizdaroglu et al., 1991, Ljungman and Hanawalt, 1992) and the vulnerability/insufficiency of the mitochondrial repair pathways (Bohr, 2002, Graziewicz et al., 2002). As a consequence, oxidative damage to mtDNA may lead to a loss of membrane potential, reduced ATP synthesis and cell death (Van Houten et al., 2006). An increasing amount of evidence suggests that oxidative stress and oxidative damage to mtDNA are linked to either the primary or secondary pathophysiologic mechanisms of multiple acute and chronic human diseases, such as atherosclerosis, neurodegeneration, and cancer (Wallace, 1999, Cohen and Tong, 2010, Galasko and Montine, 2010, Ziech et al., 2010).
In recent years, the traditional therapeutic approach to these diseases has increasingly opened up to the contribution of antioxidant supplements, especially those from natural sources which have a higher bioavailability and therefore higher protective efficacy than synthetic antioxidants (Berger, 2005, Fusco et al., 2007, Herrera et al., 2009). Focusing our attention on natural and bioavailable sources of antioxidants, we took into consideration a nutritional supplement, namely CELLFOOD® (CF) (Nu Science Corporation, CA, USA), a highly-concentrated proprietary formulation containing 78 ionic/colloidal trace elements and minerals combined with 34 enzymes and 17 amino acids, all suspended in a solution of deuterium sulfate (Dyer, 2000).
Preliminary evidence make CF potentially interesting as a natural supplement for the antioxidant protection against oxidative stress-related damage. First, the efficacy of CF has been evidenced in the treatment of fibromyalgia (Nieddu et al., 2007), a chronic pain syndrome without an effective cure (Smith and Barkin, 2010). Among the different hypotheses for its ethiopathophysiology, oxidative stress, generated by mitochondrial dysfunction, is one of the possibilities (Pieczenik and Neustadt, 2007, Cordero et al., 2010), suggesting that supplementation with antioxidants might be important in modulating the effects of ROS in the fibromyalgic syndrome. Accordingly, it has been demonstrated that the oral supplementation of CF for a period of six months significantly improves fibromyalgia symptoms and health-related quality of life of fibromyalgic patients with respect to placebo, thus sustaining the role of CF as a valuable source of antioxidants (Nieddu et al., 2007).
Second, the efficacy of CF has been evidenced in professional athletes (Milić and Djordjević, 2009). It is well known that during intense exercise, bodily oxygen consumption is greatly increased and more ROS are produced in vivo due to increased cellular metabolism and to white blood cell activation (Santos-Silva et al., 2001). If not readily neutralized, free radicals alter the permeability and functionality of muscle cell membranes thus causing a decline in performance and a slower recovery; at the same time, an anemic state may occur due to erythrocyte oxidative hemolysis (Robinson et al., 2006). Interestingly, it has been demonstrated that CF supplementation has a positive effect on the adaptation/training process and sport performance in professional cyclists (Milić and Djordjević, 2009), possibly through mechanisms involving an antioxidant protection against exercise-related oxidative damages.
With the aim of filling the existing gap in literature on the mechanisms underlying the protective role of this nutritional supplement, in the present study we investigate the antioxidant properties of CF in vitro by evaluating its protection against three pathophysiologically relevant oxidants such as hydrogen peroxide (H2O2), peroxyl radicals (ROO) and hypochlorous acid (HOCl). It is well known that, at physiological levels (1–10 μM extracellular), H2O2 enhances cell proliferation and has a signaling role; on the contrary, at higher concentrations, H2O2 induces oxidative stress, DNA oxidation and damage, and consequent mutagenesis and apoptosis (Song et al., 2007). Similarly, peroxyl radicals are important intermediates contributing significantly to free radical mediated oxidative stress, having the ability to induce chain reactions and lipoperoxidation, causing structural changes and making the membrane lose its integrity (Kannan and Jain, 2000). Finally, HOCl is a highly reactive biological oxidant playing an important role in both bacterial cell killing and inflammatory tissue injury by neutrophils (Winterbourn, 2002). Excessive production of HOCl (up to 200 μM in pathological conditions) has toxic effects; in fact, HOCl is able to penetrate cell membranes and to react with a wide range of target molecules (lipids, proteins, and DNA). Reduced glutathione is one of the preferred biological substrates of HOCl (Winterbourn and Brennan, 1997).
With this in mind, the protective effect of CF against oxidative damage was investigated in different model systems by choosing both biomolecules (glutathione and DNA) and circulating cells (erythrocytes and lymphocytes) as targets of oxidation.
CELLFOOD® (liquid) was kindly provided by Eurodream (La Spezia, Italy) and stored at room temperature (CF is stable for several years in these conditions); Lymphoprep™ was purchased from Fresenius Kabi (Oslo, Norway); 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) as generator of peroxyl radicals was obtained from Trimital (Milan, Italy); 2′,7′-dichlorofluorescin diacetate (DCFH-DA), 5,5-dithio-bis-2-nitrobenzoic (DTNB), glutathione (GSH) and sodium hypochlorite (NaOCl) from Sigma–Aldrich
The evaluation of CF ferric reducing ability by the BAP test, as a measure of its total antioxidant power, revealed that CF had a BAP value equal to 65,205 ± 1676 μM.
In the experiments using GSH as the target of oxidation, we found that H2O2, AAPH, and HOCl caused a strong oxidation of the GSH thiol group thus leading to a significant reduction in GSH concentration in the reaction mixture (approximately a reduction of 70% with respect to control was observed, data not shown). When oxidation
In this study, the in vitro antioxidant properties of CELLFOOD® were investigated for the first time. The first analysis conducted on CF (liquid) involved an evaluation of its ferric reducing ability as a measure of its total antioxidant power. As a result, we found that CF had a BAP value of approximately 65,000 μM; this means a very high antioxidant capacity considering that human plasma from healthy subjects normally has a BAP value ranging from 2200 to 4000 μM (Martarelli and Pompei, 2009).
In the present study we have clearly evidenced that CELLFOOD® is a nutritional supplement that furnishes effective antioxidant protection against pathophysiologically relevant oxidant agents. The in vitro protection of cells and biomolecules against free radical attacks suggests that CF might be a valuable coadjuvant in the prevention and treatment of various physiological and pathological conditions related to oxidative stress, from aging to sport anemia, from fibromyalgia to neurodegeneration
The authors declare that there are no conflicts of interest.
We thank Dr. Francesca Carducci for advice on the English language.
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Review article
Comparative reactivity of myeloperoxidase-derived oxidants with mammalian cells
Rayner B.S., …, Hawkins C.L.
Free Radical Biology and Medicine • Volume 71 • 2014
Citation Excerpt:It is evident from these studies that exposure of RBCs to physiologically high concentrations of HOCl leads to a rapid loss of membrane integrity, resulting in hemolysis. Studies employing increasing concentrations of HOCl from 0.125 [139], 0.5 [138], 1 [125], through to 5 mM [125,140] all observe significant increases in the presence of extracellular Hb within 10–15 min of exposure to HOCl. Furthermore, exposure of RBCs to HOCl in the range of 2–8 nmol/107 cells (equivalent to a concentration of 0.25–1 mM) for the longer period of 8 h resulted in a dose-dependent increase in the extent of hemolysis from 5% of the cell population at the lower dose, increasing to 30% with 0.5 mM, through to complete hemolysis upon exposure to 1 mM HOCl [141].
Myeloperoxidase is an important heme enzyme released by activated leukocytes that catalyzes the reaction of hydrogen peroxide with halide and pseudo-halide ions to form various hypohalous acids. Hypohalous acids are chemical oxidants that have potent antibacterial, antiviral, and antifungal properties and, as such, play key roles in the human immune system. However, increasing evidence supports an alternative role for myeloperoxidase-derived oxidants in the development of disease. Excessive production of hypohalous acids, particularly during chronic inflammation, leads to the initiation and accumulation of cellular damage that has been implicated in many human pathologies including atherosclerosis, neurodegenerative disease, lung disease, arthritis, inflammatory cancers, and kidney disease. This has sparked a significant interest in developing a greater understanding of the mechanisms involved in myeloperoxidase-derived oxidant-induced mammalian cell damage. This article reviews recent developments in our understanding of the cellular reactivity of hypochlorous acid, hypobromous acid, and hypothiocyanous acid, the major oxidants produced by myeloperoxidase under physiological conditions.
Singhal K.G., Gupta G.D.
Asian Pacific Journal of Tropical Medicine • Volume 5 • 2012
To investigate the antioxidant and hepatoprotective activity of methanolic flower extract of Nerium oleander against CCl4-induced hepatotoxicity in rats.
In vitro antioxidant activity of methanolic extract of flowers of Nerium oleander (MENO–F) was evaluated by various assays, including reducing power, lipid peroxidation, DPPH, ABTS, superoxide anion, hydroxyl radicals and metal chelation. The hepatoprotective and in vivo antioxidant activity of MENO-F were evaluated against CCl4–induced hepatic damage in rats. The MENO-F at dose of 100, 200 and 400 mg/kg were administered orally once daily for seven days. Serum enzymatic levels of serum glutamate oxaloacetate transaminase (AST), serum glutamate pyruvate transaminase (ALT), serum alkaline phosphatase (ALP) and total bilirubin were estimated along with estimation of superoxide dismutase (SOD) and malondialdehyde (MDA) levels in liver tissues. Further histopathological examination of the liver sections was carried out to support the induction of hepatotoxicity and hepatoprotective efficacy.
The extract showed potent activities on reducing power, lipid peroxide, DPPH, ABTS, superoxide anion, hydroxyl radical and metal chelation. The substantially elevated serum enzymatic levels of AST, ALT, ALP and total bilirubin were found to be restored towards normalization significantly by the MENO-F in a dose dependent manner with maximum hepatoprotection at 400 mg/kg dose level. The histopathological observations supported the biochemical evidences of hepatoprotection. Elevated level of SOD and decreased level of MDA further strengthen the hepatoprotective observations.
The results of the present study strongly reveal that MENO-F has potent antioxidant activity and hepatoprotective activity against CCl4-induced hepatic damage in experimental animals.
Sasipriya G., Siddhuraju P.
Food and Chemical Toxicology • Volume 50 • 2012
Citation Excerpt:
The antioxidant activity (AA) of the extracts was evaluated in terms of bleaching of β-carotene using the following formula: AA = [1 − (A0–At)/(A′0–A′t)] × 100, where A0 and A′0 are absorbance of values measured at zero time of the incubation for test sample and control, respectively and At and A′t are absorbances measured in the test sample and control, respectively, after incubation for 120 min. The DNA protection assay was performed using pBR 322 plasmid DNA as described by Benedetti et al. (2011) with some modifications. The reaction mixture contained 2 μL plasmid DNA (0.1 mg/mL) in phosphate buffered saline (pH 7.4) and sample extracts.
The present study is proposed to determine the antioxidant activity of raw and processed samples of underutilized legumes, Entada scandens seed kernel and Canavalia gladiata seeds. The indigenous processing methods like dry heating, autoclaving and soaking followed by autoclaving in different solutions (plain water, ash, sugar and sodium bicarbonate) were adopted to seed samples. All other processing methods than dry heat showed significant reduction in phenolics (2.9–63%), tannins (26–100%) and flavonoids (14–67%). However, in processed samples of E. scandens, the hydroxyl radical scavenging activity and β-carotene bleaching inhibition activity were increased, whereas, 2,2-azinobis (3-ethyl benzothiazoline-6-sulfonic acid) diammonium salt (ABTS+), ferric reducing antioxidant power (FRAP), metal chelating and superoxide anion scavenging activity were similar to unprocessed ones. In contrary, except dry heating in C. gladiata, all other processing methods significantly (P < 0.05) reduced the 2,2′-diphenyl-1-picryl-hydrazyl (DPPH) (20–35%), ABTS+ (22–75%), FRAP (34–74%), metal chelating (30–41%), superoxide anion radical scavenging (8–80%), hydroxyl radical scavenging (20–40%) and β-carotene bleaching inhibition activity (15–69%). In addition, the sample extracts of raw and dry heated samples protected DNA damage at 10 μg. All processing methods in E. scandens and dry heating in C. gladiata would be a suitable method for adopting in domestic or industrial processing.
Catalani S., …, Benedetti S.
Plos One • Volume 12 • 2017 • Article e0172138
Essential oils from the aerial parts (leaves, twigs and berries) of Pistacia lentiscus (PLEO) have been well characterized for their antibacterial and anti-inflammatory properties; however, poor information exists on their potential anticancer activity.
Increasing concentrations of PLEO (0.01–0.1% v/v, 80–800 μg/ml) were administered to a wide variety of cultured cancer cells from breast, cervix, colon, liver, lung, prostate, and thyroid carcinomas. Fibroblasts were also included as healthy control cells. Cell viability was monitored by WST-8 assay up to 72 hours after PLEO administration. The intracellular formation of reactive oxygen species (ROS), the induction of apoptosis, and the enhancement of chemotherapeutic drug cytotoxicity by PLEO were further investigated in the most responsive cancer cell line.
A dose-dependent reduction of tumor cell viability was observed upon PLEO exposure; while no cytotoxic effect was revealed in healthy fibroblasts. FTC-133 thyroid cancer cells were found to be the most sensitive cells to PLEO treatment; accordingly, an intracellular accumulation of ROS and an activation of both the extrinsic and intrinsic apoptotic pathways were evidenced in FTC-133 cells after PLEO administration. Furthermore, the cytotoxic effect of the antineoplastic drugs cisplatin, 5-fluorouracil and etoposide was enhanced in PLEO-exposed FTC-133 cells.
Taking into account its mode of action, PLEO might be considered as a promising source of natural antitumor agents which might have therapeutic potential in integrated oncology.
Rationale for the Successful Management of EDTA Chelation Therapy in Human Burden by Toxic Metals
Ferrero M.E.
Biomed Research International • Volume 2016 • 2016 • Article 8274504
Reactive oxygen species a double-edged sword for mesothelioma
Benedetti S., …, Galati R.
Oncotarget • Volume 6 • 2015
