The production of hydrogen peroxide is not a common mechanism by which olive oil phenols induce apoptosis on HL60 cells Roberto Fabiani a,⇑ , Patrizia Rosignoli a , Angelo De Bartolomeo a , Raffaela Fuccelli a , Maurizio Servili b , Guido Morozzi a a Dipartimento di Specialità Medico-Chirurgiche e Sanità Pubblica, Sezione di Epidemiologia Molecolare ed Igiene Ambientale, Italy b Dipartimento di Scienze Economico-Estimative e degli Alimenti, Sezione di Tecnologie e Biotecnologie Alimentari, Università degli Studi di Perugia, Italy article info Article history: Received 3 May 2010 Received in revised form 23 August 2010 Accepted 12 October 2010 Keywords: Polyphenols Hydroxytyrosol Olive oil Apoptosis Hydrogen peroxide abstract We have recently demonstrated that hydroxytyrosol (3,4-DHPEA), the most representative olive oil phenol, induces apoptosis on HL60 cells through the production of considerable amount of extracellular hydrogen peroxide (H 2 O 2 ). The aims of the present investigation were first to assess the ability of differ- ent phenolic compounds to both produce extracellular H 2 O 2 and induce apoptosis on HL60 cells, and second to elucidate whether the pro-apoptotic activity was mediated by the production of H 2 O 2 in the cell culture medium. Based on the results phenols can be classified as follows: (1) those which were not able to induce both apoptosis and H 2 O 2 accumulation (tyrosol, homovanillic alcohol and proto- catechuic, o-coumaric, vanillic, homovanillic, ferulic and syringic acids); (2) those which showed a pro-apoptotic activity mediated, at least in part, by the production of H 2 O 2 , as evidenced by the ability of catalase to inhibit apoptosis (3,4-DHPEA, dopamine, 3,4-dihydroxyphenylacetic, 3,4-dihydroxy-hydro- cinnamic, caffeic and gallic acids); and (3) those which induced apoptosis without the involvement of H 2 O 2 (the secoiridoid derivatives of both hydroxytyrosol and tyrosol). Oleuropein showed a peculiar behaviour since, and although it caused an abundant production of H 2 O 2 in the cell culture medium, it exerted a weak pro-apoptotic effect. From these results we may conclude that the cathecol moiety of the phenol molecule is necessary for the H 2 O 2 producing activity, and that the 3,4-DHPEA metabolism to homovanillic alcohol and homovanillic acid may significantly reduce its pro-apoptotic potential. The real in vivo meaning of the phenol-induced H 2 O 2 production remains to be investigated. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Dietary phenolic compounds, abundantly present in vegetables and fruits, have been shown to possess preventive activities to- ward chronic-degenerative diseases, such as cardiovascular dis- eases and cancer (Kris-Etherton et al., 2002). In the case of cancer, these compounds are believed to inhibit the carcinogenic process at the initiation stage by reducing the oxidative DNA dam- age through their free radical scavenger activity (Fernandez-Pen- chon, Villano, Troncoso, & Garcia-Parrilla, 2008). However, phenols are also able to slow down the cancer promotion/progres- sion by the interaction with different intracellular targets resulting in the inhibition of proliferation and the induction of apoptosis in tumour cells (Aggarwal & Shishodia, 2006). Olive oil contains dif- ferent phenolic compounds represented by phenolic acids, pheno- lic alcohols, flavonoids, secoiridoids and lignans (Servili & Montedoro, 2002). Among the phenolic alcohols, hydroxytyrosol [3,4-dihydroxyphenylethanol (3,4-DHPEA)] and tyrosol [p- hydroxyphenyl ethanol (p-HPEA)] are abundantly present in olive oil as both free compounds and linked to the dialdehydic form of elenolic acid, giving rise to the main secoiridois derivatives 3,4- DHPEA-EDA and p-HPEA-EDA, respectively (Fig. 1) (Servili & Montedoro, 2002). It is noteworthy that 3,4-DHPEA, p-HPEA and their secoiridoid derivatives, which include also oleuropein (Fig. 1), oleuropein aglycon and ligstroside aglycon, are exclusively present in olive oil (Servili & Montedoro, 2002). In the last few years, 3,4-DHPEA has been the subject of a considerable interest being deeply studied because of its strong antioxidant activity (for review see Fitò et al., 2007; Visioli, Poli, & Galli, 2002; Water- man & Lockwood, 2007). Indeed, this compound is able to effi- ciently prevent the hydrogen peroxide-induced DNA damage in a low concentration range (1–10 lM) (Fabiani et al., 2008). However, at high concentrations (100–400 lM) 3,4-DHPEA is able to induce apoptosis on different human cancer cells, such as colon carci- noma, melanoma and leukaemia HL60 cells (D’Angelo et al., 2005; Fabiani et al., 2002; Guichard et al., 2006). Interestingly, we have recently demonstrated that 3,4-DHPEA at 100 lM induces 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.10.052 ⇑ Corresponding author. Address: Dipartimento di Specialità Medico-Chirurgiche e Sanità Pubblica, Via del Giochetto, 06126 Perugia, Italy. Tel.: +39 075 5857336; fax: +39 075 5857328. E-mail address: fabirob@unipg.it (R. Fabiani). Food Chemistry 125 (2011) 1249–1255 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem