Hydroxytyrosol glucuronides protect renal tubular epithelial cells against H 2 O 2 induced oxidative damage Monica Deiana a,⇑ , Alessandra Incani a , Antonella Rosa a , Angela Atzeri a , Debora Loru a , Barbara Cabboi a , M. Paola Melis a , Ricardo Lucas b , Juan C. Morales b , M. Assunta Dessì a a Dipartimento di Biologia Sperimentale, Sezione di Patologia Sperimentale, Università degli Studi di Cagliari, Cittadella Universitaria SS 554, 09042 Monserrato, Cagliari, Italy b Department of Bioorganic Chemistry, Instituto de Investigaciones Químicas, CSIC-Universidad de Sevilla, 49 Americo Vespucio, 41092 Sevilla, Spain article info Article history: Received 16 May 2011 Received in revised form 4 July 2011 Accepted 10 July 2011 Available online 26 July 2011 Keywords: Hydrogen peroxide Hydroxytyrosol glucuronides Lipid peroxidation MAPK Oxidative stress abstract Hydroxytyrosol (2-(3 0 ,4 0 -dihydroxyphenyl)ethanol; HT), the most active ortho-diphenolic compound, present either in free or esterified form in extravirgin olive oil, is extensively metabolized in vivo mainly to O-methylated, O-sulfated and glucuronide metabolites. We investigated the capacity of three glucuro- nide metabolites of HT, 3 0 -O-b-D-glucuronide and 4 0 -O-b-D-glucuronide derivatives and 2-(3 0 ,4 0 -dihy- droxyphenyl)ethanol-1-O-b-D-glucuronide, in comparison with the parent compound, to inhibit H 2 O 2 induced oxidative damage and cell death in LLC-PK1 cells, a porcine kidney epithelial cell line. H 2 O 2 treat- ment exerted a toxic effect inducing cell death, interacting selectively within the pro-death extracellular- signal relate kinase (ERK 1/2) and the pro-survival Akt/PKB signaling pathways. It also produced direct oxidative damage initiating the membrane lipid peroxidation process. None of the tested glucuronides exhibited any protection against the loss in renal cell viability. They also failed to prevent the changes in the phosphorylation states of ERK and Akt, probably reflecting their inability to enter the cells, while HT was highly effective. Notably, pretreatment with glucuronides exerted a protective effect at the high- est concentration tested against membrane oxidative damage, comparable to that of HT: the formation of malondialdehyde, fatty acid hydroperoxides and 7-ketocholesterol was significantly inhibited. Ó 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Hydroxytyrosol (2-(3 0 ,4 0 -dihydroxyphenyl)ethanol; HT) is the most potent antioxidant among the phenolics found in extravirgin olive oil. The biological activities of HT have been explored by sev- eral groups as recently reviewed by Granados-Principal et al. [1]. Two of the most relevant reported activities are antitumor and anti-inflammatory effects. The anticancer activity seems to be a re- sult of its capacity to exert cytotoxic effects, such as induction of apoptosis, cell cycle arrest and antiproliferative effects. The anti- inflammatory and antiplatelet aggregation action of HT, together with its antiatherogenic capacity and cardioprotective effects, are important in counteracting the development of cardiovascular diseases. HT biological effects stem mainly from its free radical scaveng- ing and metal chelating properties, most probably due to its ortho-diphenolic structure, whose high antioxidant activity may be explained by the high electron donating effect of the second hydroxyl group. At the same time it also shows effects on cell sig- naling pathways and on gene expression [1]. A daily intake of 25–50 ml of extravirgin olive oil, typical of Med- iterranean countries, may supply at least 1 mg of simple phenols, free HT and tyrosol, and 8 mg of their secoiridoids derivatives, mainly oleuropein and ligstroside-aglycones [2]. However, the majority of these complex polyphenols undergo gastro-intestinal biotransformation. HT and tyrosol conjugated forms, except oleu- ropein, are rapidly hydrolysed under gastric conditions, effectively increasing the relative amount of simple phenols, HT and tyrosol, entering the small and large intestine [3]. Oleuropein is not degraded under acidic conditions and is not absorbed in the paren- tal form in the small intestine; however, once reached the large intestine, it may be subjected to rapid degradation by the colonic microflora, to yield HT [3]. The absorption of HT takes place in the small intestine and the colon [4] through a passive diffusion mechanism [5]. In the process of crossing enterocytes, HT is subjected to a classic phase I/II bio- transformation, and then to an important first pass metabolism in the liver cells. This process leads to the formation of ortho- methyl derivatives (homovanillic alcohol), glucuronide and sulfate 0009-2797/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2011.07.002 Abbreviations: HT, hydroxytyrosol; HP, fatty acid hydroperoxides; UFA, unsaturated fatty acids; 7-keto, 7-ketocholesterol; Glu1, hydroxytyrosol 3 0 -O-b-D- glucuronide; Glu2, hydroxytyrosol 4 0 -O-b-D-glucuronide; Glu3, 2-(3 0 ,4 0 -dihydroxy- phenyl)ethanol-1-O-b-D-glucuronide; ERK, extracellular-signal related kinase. ⇑ Corresponding author. Tel.: +39 070 6754126; fax: +39 070 6754032. E-mail address: mdeiana@unica.it (M. Deiana). Chemico-Biological Interactions 193 (2011) 232–239 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint