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

Abstract

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 µM cumene hydroperoxide. Wide range dose-dependence 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.

Protection by extra virgin olive oil against oxidative stress in vitro and in vivo. Chemical and biological studies on the health benefits due to a major component of the Mediterranean diet

“…Cells reached confluency after 5 days (approximately 6.0 x 10 6 cells/flask) and were kept in culture as myoblasts by continuous passages at preconfluent stages as previously reported [ 18 ]. Human leukemic monocytes, THP-1, from American Type Culture Collection (Rockville, MD, USA), were grown in suspension in RPMI-1640 medium with 10% FBS, 100 μg /ml streptomycin and 100 U/ml penicillin, in a humidified atmosphere with 5% CO 2 a t 37°C [ 19 – 20 ] THP-1 monocytes were passaged twice a week by 1:4 dilutions and re-seeded; cells from passages 7–23 were used for the experiments.…”

“…Reactive oxygen species (ROS) were measured both in L6 and THP-1 cells. The method used was a standard assay based on the intracellular fluorescent probe DCF [ 20 ]. For L6 myoblasts, the medium was discarded and cells were washed twice with 5 ml phosphate buffered saline (PBS) containing 5.0 mM glucose (PBS-glucose), at 37°C.…”

Negletein as a neuroprotectant enhances the action of nerve growth factor and induces neurite outgrowth in PC12 cells

“…In this study, the possible neuritogenic mechanism of negletein is bound to the activation of tyrosine kinase receptor (TrkA) by the NGF and downstream signaling of ERK1/2, PI3K and the other activated signaling pathways. The effect is probably due to the stimulation of kinases because of ROS production as reported for the GTPP ; as well as to the antioxidant activity for negletein . Therefore, negletein at a low micro molar range appears to be a moderate antioxidant with iron chelating properties and it moderates ROS‐mediated cell signaling enhancing effects.…”

A Tricin Derivative from Deschampsia antarctica Desv. Inhibits Colorectal Carcinoma Growth and Liver Metastasis through the Induction of a Specific Immune Response

“…Certain compounds derived from medicinal plants, such as flavonoids and isoflavonoids, have received much attention because of their protective effects observed in populations with low incidence of colorectal, breast, and prostate cancer (14). Their antitumor activity involves a number of mechanisms: flavonoids act on reactive oxygen species, on cell signal transduction pathways related to cellular proliferation by inhibiting the transcription factor NF-kB or by inducing apoptosis, and also on cell migration (26)(27)(28)(29). In particular, their capacity to inhibit tyrosine kinase activity and to suppress metalloproteinases was associated with inhibition of tumor development (30,31).…”