Protection of Cells against Oxidative Stress by Nanomolar Levels of Hydroxyflavones Indicates a New Type of Intracellular Antioxidant Mechanism

et al. (2013) Protection of Cells against Oxidative Stress by Nanomolar Levels of Hydroxyflavones Indicates a New Type of Intracellular Antioxidant Mechanism. PLoS ONE 8(4): e60796. doi:10.1371/journal.pone.0060796 Protection of Cells against Oxidative Stress by Nanomolar Levels of Hydroxyflavones Indicates a New Type of Intracellular Antioxidant Mechanism Emanuele Lombardo 0 Cristian Sabellico 0 Jan Ha jek 0 Veronika Stan kova 0 Toma s Filipsky 0 Valentina Balducci 0 Paolo De Vito 0 Stefano Leone 0 Eugenia I. Bavavea 0 Ilaria Proietti Silvestri 0 Giuliana Righi 0 Paolo Luly 0 Luciano Saso 0 Paolo Bovicelli 0 Jens Z. Pedersen 0 Sandra Incerpi 0 Luis Eduardo M. Quintas, Universidade Federal do Rio de Janeiro, Brazil 0 1 Department of Sciences, University Roma Tre , Rome , Italy , 2 Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kra love , Charles University , Prague , Czech Republic , 3 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kra love , Charles University , Prague , Czech Republic , 4 Department of Biology, University of Rome Tor Vergata , Rome , Italy , 5 Department of Medicinal Chemistry, Faculty of Pharmacy, University of Athens, Greece, 6 Department of Chemistry, Sapienza University of Rome , Rome , Italy , 7 Institute of Biomolecular Chemistry, National Research Council (CNR) , Rome , Italy , 8 Department of Physiology and Pharmacology ''Vittorio Erspamer'', Sapienza University of Rome , Rome , Italy Natural polyphenol compounds are often good antioxidants, but they also cause damage to cells through more or less specific interactions with proteins. To distinguish antioxidant activity from cytotoxic effects we have tested four structurally related hydroxyflavones (baicalein, mosloflavone, negletein, and 5,6-dihydroxyflavone) at very low and physiologically relevant levels, using two different cell lines, L-6 myoblasts and THP-1 monocytes. Measurements using intracellular fluorescent probes and electron paramagnetic resonance spectroscopy in combination with cytotoxicity assays showed strong antioxidant activities for baicalein and 5,6-dihydroxyflavone at picomolar concentrations, while 10 nM partially protected monocytes against the strong oxidative stress induced by 200 mM cumene hydroperoxide. Wide range dosedependence curves were introduced to characterize and distinguish the mechanism and targets of different flavone antioxidants, and identify cytotoxic effects which only became detectable at micromolar concentrations. Analysis of these dose-dependence curves made it possible to exclude a protein-mediated antioxidant response, as well as a mechanism based on the simple stoichiometric scavenging of radicals. The results demonstrate that these flavones do not act on the same radicals as the flavonol quercetin. Considering the normal concentrations of all the endogenous antioxidants in cells, the addition of picomolar or nanomolar levels of these flavones should not be expected to produce any detectable increase in the total cellular antioxidant capacity. The significant intracellular antioxidant activity observed with 1 pM baicalein means that it must be scavenging radicals that for some reason are not eliminated by the endogenous antioxidants. The strong antioxidant effects found suggest these flavones, as well as quercetin and similar polyphenolic antioxidants, at physiologically relevant concentrations act as redox mediators to enable endogenous antioxidants to reach and scavenge different pools of otherwise inaccessible radicals. - Funding: The financial support from the Italian Ministry for Education, University and Research, General Management for International Research, is gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The radical scavenging properties of flavonoids and similar polyphenolic compounds are due to the presence of hydroxy groups on the aromatic ring system able to donate electrons or hydrogen atoms, and sometimes also to act as chelators of redoxactive metal ions [15]. One class of flavonoids are the flavones which often are antioxidants, but flavones with methoxy groups also have other interesting biological effects. Despite the potential importance of this group of compounds relatively few studies on their intracellular antioxidant effects have been published; their use is limited by the scarce availability of most of them [6,7]. Baicalein is a flavone derived from the roots of the plant Scutellaria baicalensis, one of the most popular traditional medicine herbs used in China. It has a very potent antioxidant activity compared to most other flavonoids, causing attenuation of oxidative stress effects in cardiomyocytes, and neuroprotection and anxiolytic effects in neuronal cells, and it is a mild relaxant compound in the neuromuscular system and skeletal muscles [8 10]. The dry root of S. baicalensis also contains other flavones (in particular baicalin, wogonin and wogonoside); it has been widely used in different pathological conditions such as hyperlipemia, atherosclerosis, hypertension, and inflammatory diseases [10]. Both baicalein and S. baicalensis extracts show cytostatic activity for many types of cancer cells in culture, and are able to inhibit proliferation of different human myeloma cell lines and other types of tumors in vivo [1012]. Interestingly only little such inhibition was seen for non-tumor cells, even at high doses [13]; this low toxicity for non-tumor cells makes baicalein a very interesting antitumor drug candidate. The anti-tumor activity appears to be a multifactorial process involving several mechanisms, such as cell cycle inhibition, scavenging of superoxide, the inhibition of the redox-regulated transcription factor NF-kB, but also pro-oxidant activity causing induction of apoptosis [10]. Other natural flavones have been less characterized as to their antioxidant properties. However, it is known that such polyphenol compounds besides their protective effects due to radical scavenging also produce various side effects responsible for the cytotoxicity of many of them. How much of this toxicity depends on the high concentrations employed in vitro, and how important it may be in vivo is not clear. Their antiproliferative effect may be directly related to the general cytoxicity of these compounds, independently of their antioxidant properties; therefore their adverse effects must be examined before they are evaluated as therapeutic tools. The potential importance of baicalein and similar molecules, together with the lack of information on the effects of flavones in general, prompted us to examine the antioxidant properties of four structurally related flavones: mosloflavone, negletein, 5,6-dihydroxyflavone, and baicalein. The s (...truncated)