Raffaela Fuccelli

Effect of olive oil phenols on the production of inflammatory mediators in freshly isolated human monocytes

Recent in vitro and in vivo studies suggest that the anti-inflammatory properties of extra virgin olive oil may be involved in the prevention of chronic degenerative diseases. In this study, the ability of olive oil phenols to influence the release of superoxide anions (O2-), prostaglandin E2 (PGE2) and tumor necrosis factor α (TNFα) and the expression of cyclooxygenase2 (COX2) in human monocytes, freshly isolated from healthy donors, was investigated. O2- were measured by superoxide dismutase-inhibitable cytochrome c reduction and PGE2 and TNFα production were determined in culture medium with appropriate enzyme immunoassay kits. COX2 mRNA and protein were evaluated by quantitative reverse transcription-polymerase chain reaction and Western immunoblotting, respectively. Treatment of monocytes for 24 h with 100 μM of hydroxytyrosol (3,4-DHPEA), tyrosol (p-HPEA) and their secoiridoid derivatives (3,4-DHPEA and p-HPEA linked to the dialdehydic form of elenolic acid: 3,4-DHPEA-EDA and p-HPEA-EDA, respectively) significantly (P<.05) inhibited the production of O2(-) as follows: 3,4-DHPEA (40%,), p-HPEA (9%), 3,4-DHPEA-EDA (25%) and p-HPEA-EDA (36%). Hydroxytyrosol also considerably reduced the expression of COX2 at both the mRNA and protein level (P<.05) and caused a clear dose-dependent reduction of PGE2 released into the culture medium (45% and 71% at 50 and 100 μM, respectively, P<.05). The COX2 mRNA was also efficiently inhibited by the secoiridoids. Moreover, it was shown that hydroxytyrosol increased the monocytes TNFα production. In addition to other chemopreventive properties, these results suggest that the health effects of olive oil phenols may be related to their ability to modulate the production of pro-inflammatory molecules, a property common to non-steroidal anti-inflammatory drugs.

Genotoxic effect of bile acids on human normal and tumour colon cells and protection by dietary antioxidants and butyrate

BackgroundColorectal cancer is the second cause of death for tumour worldwide. Among the risk factors for this disease the dietary habits seem to have a pivotal role. An elevated intake of fats causes a high release in the gut lumen of bile acids that are positively correlated with colorectal cancer, since they act as detergents and proliferation promoters. Recently, it was evidenced that bile acids can also be able to induce DNA damage.Aim of the studyIn this study the genotoxicity of deoxycholic acid (DCA) and chenodeoxycholic acid CDCA) has been evaluated in human normal colonocytes derived from 60 colon biopsies and in tumour cells. The involvement of reactive oxygen species (ROS) and the oxidative DNA damage was assessed. In addition, the protective effect exerted by both two well-known antioxidants commonly present in the diet, β-carotene and α-tocopherol, and butyrate which is known to be involved in the regulation of several cellular functions, has also been tested.MethodsThe DNA damage was evaluated by the “comet assay” or single cell gel electrophoresis (SCGE) both in its conventional use and by the Endonuclease III modified method, which allow to detect the presence of oxidized pyrimidines.ResultsBile acids (CDA and CDCA) resulted genotoxic on both normal and tumour human colon cells. The inclusion of the endonuclease III digestion step in the comet assay demonstrated that bile acids induced an oxidative DNA damage. In addition, treatment of colonocytes with bile acids in the presence of the antioxidants (β-carotene, α-tocopherol) and Na-butyrate caused a reduction of DNA damage.ConclusionOur results suggest that bile acids may be involved in the tumour initiation by inducing a DNA oxidative damage, and so add further evidences to the preventive properties of antioxidants present in the Mediterranean diet.

Production of hydrogen peroxide is responsible for the induction of apoptosis by hydroxytyrosol on HL60 cells

Hydroxytyrosol [3,4-dihydroxyphenylethanol (3,4-DHPEA)], a phenolic compound found exclusively in olive oil, exerts growth-suppressive and pro-apoptotic effects on different cancer cells. Although some molecular mechanisms involved in the pro-apoptotic activity of 3,4-DHPEA have been proposed, the initial stress signals responsible of this phenomenon are not known. Our aim was to assess the involvement of reactive oxygen species as mediators of apoptosis induced by 3,4-DHPEA on HL60 cells. Apoptosis was determined by analyzing the nuclear fragmentation by both fluorescence microscopy and flow cytometry. The externalization of phosphatidylserine was evidenced using an Annexin V-FITC kit. The concentration of H(2)O(2) in the culture medium was measured by the ferrous ion oxidation-xylenol orange method. The pro-apoptotic effect of 3,4-DHPEA (100 muM) was prevented by N-acetyl-cysteine, ascorbate, and alpha-tocopherol. Catalase suppressed the 3,4-DHPEA-induced apoptosis, while the Fe(II)-chelating reagent o-phenantroline showed no effect, suggesting the involvement of H(2)O(2 )but not of OH(*). Indeed, 3,4-DHPEA caused accumulation of H(2)O(2) in the culture medium. Tyrosol (p-hydroxyphenylethanol) and caffeic acid, compounds structurally similar to 3,4-DHPEA but not able to generate H(2)O(2), did not induce an appreciable apoptotic effect. This is the first study demonstrating that apoptosis induction by 3,4-DHPEA is mediated by the extracellular production of H(2)O(2).

Influence of Cultivar and Concentration of Selected Phenolic Constituents on the in Vitro Chemiopreventive Potential of Olive Oil Extracts

One of the main olive oil phenolic compounds, hydroxytyrosol (3,4-DHPEA), exerts in vitro chemopreventive activities (antiproliferative and pro-apoptotic) on tumor cells through the accumulation of H(2)O(2) in the culture medium. However, the phenol composition of virgin olive oil is complex, and 3,4-DHPEA is present at low concentrations when compared to other secoiridoids. In this study, the in vitro chemopreventive activities of complex virgin olive oil phenolic extracts (VOO-PE, derived from the four Italian cultivars Nocellara del Belice, Coratina, Ogliarola, and Taggiasca) were compared to each other and related to the amount of the single phenolic constituents. A great chemopreventive potential among the different VOO-PE was found following this order: Ogliarola > Coratina > Nocellara > Taggiasca. The antiproliferative and pro-apoptotic activities of VOO-PE were positively correlated to the secoiridoid content and negatively correlated to the concentration of both phenyl alcohols and lignans. All extracts induced H(2)O(2) accumulation in the culture medium, but this phenomenon was not responsible for their pro-apoptotic activity. When tested in a complex mixture, the olive oil phenols exerted a more potent chemopreventive effect compared to the isolated compounds, and this effect could be due either to a synergistic action of components or to any other unidentified extract constituent.

Genotoxicity of alkene epoxides in human peripheral blood mononuclear cells and HL60 leukaemia cells evaluated with the comet assay.

Volatile organic compounds (VOCs) exert their carcinogenic activity through the production of epoxide metabolites. Because of their high reactivity some epoxides are also produced in the chemical industry for the synthesis of other compounds. Therefore, human exposure to VOCs epoxides does occur and may be an important human health concern. In this study, the in vitro genotoxic potential of epoxides originating from 1,3-butadiene (3,4-epoxy-1-butene: EB; 1,2:3,4-diepoxybutane: DEB), isoprene (3,4-epoxy-2-methyl-1-butene: IO), styrene (styrene-7,8-oxide: SO), propylene (propylene oxide: PO) and 1-butene (1,2-epoxy-butane: BO) in human peripheral blood mononuclear cells (PBMCs) and promyelocytic leukaemia cells (HL60) was measured with the comet assay (single-cell gel electrophoresis, SCGE). The effect of inclusion of foetal calf serum (FCS, 5%) in the cell-culture medium and different durations of exposure (2h, 24h) were also investigated. All epoxides tested produced DNA damage in a concentration range that did not reduce cell viability. HL60 cells were more resistant than PBMCs to the DNA damage induced by the different epoxides. With the exception of IO, the treatment for 24h resulted in an increase of DNA damage. FCS slightly protected PBMCs from the genotoxic effects induced by IO and BO, whilst no such effect was noted for the other compounds. Overall, the dose-dependent effects that were seen allowed us to define a genotoxicity scale for the different epoxides as follows: SO>EB>DEB>IO>PO>BO, which is in partial agreement with the International Agency for Research on Cancer (IARC) classification of the carcinogenic hazards.

The hydroxytyrosol-dependent increase of TNF-α in LPS-activated human monocytes is mediated by PGE2 and adenylate cyclase activation

An accurate regulation of PGE2 and TNF-α production is an important event for a physiological inflammation process. We have recently reported that in LPS-activated human monocytes hydroxytyrosol, the main phenol present in extra virgin olive oil reduced both the COX-2 gene expression and PGE2 secretion while it increased the TNF-α accumulation in the culture medium. Here we have investigated whether these effects were related to each other, clarifying the possible mechanisms involved. We found that hydroxytyrosol (100 μM) increased the TNF-α mRNA level in LPS-activated human monocytes as evaluated by both RT-PCR and real time PCR (qPCR). Exogenous PGE2 reduced both TNF-α mRNA and TNF-α secretion (EIA assay) while the activation of adenylate cyclase by forskolin decreased only the TNF-α secretion but did not influence the TNF-α mRNA level. Acting similarly to non steroidal anti-inflammatory drugs (NSAIDs), the hydroxytyrosol could be used to develop innovative drugs for the control of inflammation and immune response. The decrease of TNF mediated by forskolin, moreover, could suggest that the pharmacological regulation of cAMP production may represent a strategy to control the side effects of NSAIDs.

Preventive Activity of Olive Oil Phenolic Compounds on Alkene Epoxides Induced Oxidative DNA Damage on Human Peripheral Blood Mononuclear Cells

The aim of this study was to investigate the ability of epoxides of styrene (styrene-7,8-oxide; SO) and 1,3-butadiene (3,4-epoxy-1-butene; 1,2:3,4:-diepoxybutane) to cause oxidative stress and oxidative DNA damage on human peripheral blood mononuclear cells (PBMCs) and whether a complex mixture of olive oil phenols (OOPE) could prevent these effects. The DNA damage was measured by the single-cell gel electrophoresis (SCGE; comet assay). We found that the DNA damage induced by alkene epoxides could be prevented by N-acetyl-cysteine (10 mM) and catalase (100 U/ml). Alkene epoxides caused a significant (P < 0.05) increase of both peroxide concentration in extra- and intracellular environment and formamidopyrimidine DNA glycosylase (FPG)- and Endonuclease III (ENDO III)-sensitive sites in PBMCs, demonstrating the presence of oxidized bases. OOPE (1 μg of total phenols/ml) was able to prevent the alkene epoxide induced DNA damage both after 2 and 24 h of incubation. In addition, OOPE completely inhibited the SO-induced intracellular peroxide accumulation in PBMCs and prevented the oxidative DNA damage induced by SO, as evidenced by the disappearance of both FPG- and ENDO III-sensitive sites. This is the first study demonstrating the ability of OOPE to prevent the DNA damage induced by alkene epoxides providing additional information about the chemopreventive properties of olive oil.