| Aim:Numerous epidemiological data have demonstrated an association between a diet rich in antioxidants, such as the"Mediterranean diet,"and a lower incidence of several diseases, such as cancers, atherosclerosis and coronary heart disease. Olive oil is the most important food of"Mediterranean diet,"and can modulate the diseases from studies in vivo and in vitro. Although the protective effect of such a diet is likely to be multifactorial, there is consistent evidence for an antioxidant activity of some selected polyphenolic compounds from extra virgin olive oil. Hydroxytyrosol (HT), an olive phenolic, chemiclally named (3, 4-dihydroxyphenyl) ethanol, is hydrosoluble and liposoluble moelcule. HT is an efficient scavenger of free radicals, which prevents the autooxidation of polyunsaturated fatty acid. The free radical scavenging activity of HT is higher than synthetic and natural antioxidant and such as vitamin E, vitamin C and butylated hydroxytoluenez (BHT).Environmental pollution includes water pollution, food contamination and air pollution, in which effects of pollutants in food on the health are not only extensive but also direct and cause widespread concern. In recent years, there are so many food contaminations that caused social panic about risk of cancer, such as Sudan I, Teflon, and acrylamide (AA) and so on. These potential carcinogenicity substances have been paid to attentions.The International Agency for Research on Cancer (IARC) assessed Sudan I as a Group 3 carcinogen. AA is neurotoxic in humans and laboratory animals, and was classified as"probably carcinogenic to humans"(Group 2A carcinogen) by a working group of the IARC. This might represent a potential threat to public health. The liver is not only the target site of Sudan I and AA metabolism but also the site of phenolic compound metabolism. HepG2 cells were used as the experimental system in vitro to investigate the chemoprotective effect of HT on the genotoxicity induced by Sduan I and AA in our study. The HepG2 cell line retained many of the functions of normal liver cells and expresses the activities of several phases I and II xenobiotic metabolizing enzymes that play key roles in the activation and/or detoxification of DNA-reactive carcinogens. It has been shown to be a suitable system for genotoxicity testing.A recent report also indicates that HT could reduce vascular cell adhesion molecule-1 mRNA expression by blocking the activation of transcription factors nuclear factor-kappaB (NF-κB) and activator protein-1. It has been demonstrated that natural and synthetic antioxidants inhibit pro-infla- mmatory gene expression regulated by transcription factors, including NF-κB, signal transducer and activator of transcription-1α(STAT-1α) and interferon regulatory factor-1(IRF-1). These transcription factors are dependent on the intracellular redox state. We selected THP-1 cells stimulated by LPS to study the anti-inflammatory effect of HT and the possible mechanisms.Methods: (1) Methods of HepG2 cells line as test system were as follows. The single cell gel electrophoresis assay (SCGE) in addition to the micronucleus test (MNT) to study the genotoxic effects was performed. The cell viability was examined using the methyl thiazol tetrazolium bromide (MTT) assay. In order to clarify the underlying mechanisms we measured the intracellular ROS formation using 2, 7-dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe and intracellular glutathione(GSH) level by fluorometric methods. The levels of oxidative DNA damage and lipid peroxidation were estimated by immunocytochemistry analysis of 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS), respectively. The rate-limiting enzyme in GSH synthesis is gamma-glutamylcysteine synthetase (γ-GCS), and western blot forγ-GCS was applied in present study.(2) The THP-1 cell was stimulated by LPS as the inflammtory model. ELISA was used to detect the level of tumor necrosis factor-α(TNF-α). The gene expression of TNF-α, inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) was measured by RT-PCR and the protein expression of iNOS and COX-2 was estimated by Western blot. To futher study the the relation between the anti-inflammtory effect of HT and intracellular redox state, intracellular GSH andγ-GCS were measured.Results: (1) The chemoprotective effects of HT on the genotoxicity induced by Sudan I and AA were as follows. We found that HepG2 cells treated with 100 uM Sudan I resulted in serious DNA strand breaks. In contrast, the DNA damage was significantly reduced in cells pretreatment with 25-100 uM HT in a concentration-dependent manner. The results of MNT showed that HepG2 cells treated with 100 uM Sduan I could induce the decrease of GSH and the increase of ROS, intracellular TBARS level and the increase of 8-OHdG expression. Pretreatment with HT could increase the level of GSH and decrease the level of ROS, TBARS and 8-OHdG in a concentration-dependent manner. Moreover, high dose of HT (100μM) could completely inhibit the increase the levels of ROS, TBARS and 8-OHdG. Pretreatment with HT could inhibit the cytotoxicity induced by 5mM and 10mM AA. The SCGE results showed that HepG2 cells treated with 10mM resulted in serious DNA damage. Pretreatment with doses of HT for 30 min then exposed to 10mM inhibited AA-induced DNA damage in a concentration-dependent manner. Frequencies of micronuclei significantly increased in HepG2 cells after treatment with 2.5 mM AA for 24 h. Pretreatment with doses of HT for 30 min decrease the frequencies of MN in a concentration-independent manner. Furthermore, HT was able to reduce intracellular ROS formation and attenuate GSH depletion caused by AA in a concentration-dependent manner. The futher study showed that 25μM HT enhanced the expression ofγ-GCS in HepG2 cells treated with 10 mM AA using immmnoblotting.(2) The effects of HT on the inflammation stimulated by LPS were as follows. HT could significantly decrease the increase of TNF-αlevel stimulated by LPS and inhibit the increases of iNOS and COX-2 gene expression stimulated by LPS. HT also could significantly decrease the levels of increase of iNOS and COX-2 protein expression stimulated by LPS. The level of GSH in THP-1 cells stimulated by LPS was significantly decreased and the level ofγ-GCS was significantly increased as compared to cells without LPS, and pretreatment with HT could increase the level of GSH and enhanced the level ofγ-GCS expression in a concentration-dependent manner.Conclusions: In the study, we are first to investigate the chemoprotective of HT on genotoxicity induced by Sudan I and AA in HepG2 cells. We found HT could decrease the genotoxicity induced by Sudan I and AA in HepG2 cells. Moreover, we found that HT could modulate the redox state and prevent the oxidative damage by decreasing the level of ROS and increasing the level of GSH to attenuate the the genotoxicity in HepG2 cells induced by Sudan I and AA. In addition, HT could inhibit the inflammtory response in THP-1 cells stimulated by LPS. It suggested that HT could inhibit inflammtion in THP-1 cells stimulated by LPS through decreasing the gene expression of the inflammtion-relative cytokines, which was related to increase of GSH and the enhancement ofγ-GCS expression.
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