Quercetin protects human hepatoma HepG2 against oxidative stress induced by tert-butyl hydroperoxide

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Abstract

Flavonols such as quercetin, have been reported to exhibit a wide range of biological activities related to their antioxidant capacity. The objective of the present study was to investigate the protective effect of quercetin on cell viability and redox status of cultured HepG2 cells submitted to oxidative stress induced by tert-butyl hydroperoxide. Concentrations of reduced glutathione and malondialdehyde, generation of reactive oxygen species and activity and gene expression of antioxidant enzymes were used as markers of cellular oxidative status. Pretreatment of HepG2 with 10 μM quercetin completely prevented lactate dehydrogenase leakage from the cells. Pretreatment for 2 or 20 h with all doses of quercetin (0.1–10 μM) prevented the decrease of reduced glutathione and the increase of malondialdehyde evoked by tert-butyl hydroperoxide in HepG2 cells. Reactive oxygen species generation induced by tert-butyl hydroperoxide was significantly reduced when cells were pretreated for 2 or 20 h with 10 μM and for 20 h with 5 μM quercetin. Finally, some of the quercetin treatments prevented the significant increase of glutathione peroxidase, superoxide dismutase, glutathione reductase and catalase activities induced by tert-butyl hydroperoxide. Gene expression of antioxidant enzymes was also affected by the treatment with the polyphenol. The results of the biomarkers analyzed clearly show that treatment of HepG2 cells in culture with the natural dietary antioxidant quercetin strongly protects the cells against an oxidative insult.

Introduction

Dietary antioxidants, including polyphenolic compounds, are considered beneficial because of their potential protective role in the pathogenesis of multiple diseases associated to oxidative stress such as cancer, coronary heart disease and atheroschlerosis. Flavonoids comprise a large group of naturally occurring low molecular weight polyphenolic compounds that are present in all plants (Bravo, 1998). Flavonoids, and specifically flavonols such as quercetin, have been reported to exhibit a wide range of biological activities (Hertog and Holland, 1996), including anticarcinogenic, anti-inflammatory and antiviral actions. In addition, they inhibit lipid peroxidation, platelet agregation, capillary permeability and the activity of enzyme systems including lipoxygenase (Aherne and O'Brien, 1999). The flavonoids exert these effects as antioxidants, chelators of divalent cations and free radical scavengers and thus may be involved in preventing free radical mediated cytotoxicity and lipid peroxidation which are associated with cell aging and chronic diseases (Middelton, 1996, Mora et al., 1990).

The flavonoid used in this study, quercetin, is one of the most common dietary flavonols with a well characterized in vitro antioxidant activity. It is found in fruits, vegetables, tea, wine, nuts, seeds and represents an integral part of the human diet. The average daily Western diet contains between 100 mg and 1000 mg total flavonoids, of which approximately 23 mg has been estimated as the average intake of flavonols and flavones in the Dutch population (Hertog and Holland, 1996). Quercetin is the predominant flavonol found in foods and intakes of between 6 and 31 mg per day have been reported (Hertog et al., 1995). Absorption of these compounds through the gastrointestinal tract renders measurable amounts of blood polyphenols which may affect the biochemistry of cells from different tissues, in particular the liver.

The study of the effect of dietary polyphenols on the regulation of antioxidant defense mechanisms at the cellular level may benefit from the use of an established cell culture line. Human hepatoma HepG2, a well differentiated transformed cell line, is a reliable model, easy to culture, well characterized and widely used for biochemical and nutritional studies where many antioxidants and conditions can be assayed with minor interassay variations (Alía et al., in press, Alía et al., 2005). In addition, steady-state functioning of the antioxidant defenses in human hepatoma HepG2 is relatively higher than that in hepatocytes and other nontransformed cells, therefore, variations of responses to different conditions are more easily detected (Alía et al., in press, Alía et al., 2005, Murakami et al., 2002a, Murakami et al., 2002b, Chen et al., 2002).

The objective of the present study was to investigate the potential protective effect of different quercetin concentrations against oxidative stress induced by tert-butyl hydroperoxide (t-BOOH) in HepG2 cells in culture. Cell integrity and several markers of oxidative damage were used to estimate the effect of quercetin in cell survival and the response of the antioxidant system of HepG2 to t-BOOH. Concentration of reduced glutathione (GSH); determination of malondialdehyde (MDA) as marker of lipid peroxidation; generation of reactive oxygen species (ROS); evaluation of the activity of antioxidant enzymes: catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD) and glutathione reductase (GR) and the gene expression of CAT, GPx and SOD were measured.

Section snippets

Cell culture and quercetin treatment

Human hepatoma HepG2 cells initially isolated from a liver biopsy in a 15-year-old Caucasian male (Aden et al., 1979) were a gift from Dr. Paloma Martin-Sanz (Instituto de Bioquimica, CSIC, Madrid, Spain). This cell line was grown in a humidified incubator containing 5% CO2 and 95% air at 37 °C. They were grown in DMEM F-12 medium from Biowhitaker (Innogenetics, Madrid, Spain), supplemented with 2.5% Biowhitaker fetal bovine serum (FBS) and 50 mg/L of each of the following antibiotics:

Cytotoxicity

Lactate dehydrogenase (LDH) leakage was used as an indicator of cytotoxicity. In our experimental conditions, a 3 h treatment with 200 μM t-BOOH evoked a great increase in LDH activity in the cell culture medium indicating cell damage in HepG2 (Fig. 1a). Pretreatment for 2 h of HepG2 cultures with 0.1–5 μM quercetin greatly reduced cell damage. Concentrations of quercetin of 5 μM decreased cell toxicity almost to control values (t-BOOH-untreated cells). A higher dose of quercetin (10 μM)

Discussion

There is considerable current interest in the cytoprotective effects of natural antioxidants against oxidative stress and the different defense mechanisms involved. This study demonstrates that quercetin, a common dietary flavonol with a high in vitro antioxidant activity, has the ability to protect the cell against an oxidative insult by modulating ROS generation, reduced glutathione concentration, MDA production and the main antioxidant enzymes in the cell.

Through the well documented

Acknowledgments

This work was supported by the grant AGL2000-1314 from the Spanish Ministry of Science and Technology (CICYT). S. Ramos has a contract from the Ramon y Cajal program from the Ministerio de Educación y Ciencia. R. Mateos is a postdoctoral fellow from the Ministerio de Educación y Ciencia.

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