Human hepatic cell uptake of resveratrol: involvement of both passive diffusion and carrier-mediated process

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Abstract

This work reports significant advances on the transport in hepatic cells of resveratrol, a natural polyphenol with potent protective properties. First, we describe a new simple technique to qualitatively follow resveratrol cell uptake and intracellular distribution, based on resveratrol fluorescent properties. Second, the time-course study and the quantification of 3H-labelled resveratrol uptake have been performed using human hepatic derived cells (HepG2 tumor cells) and hepatocytes. The temperature-dependence of the kinetics of uptake as well as the cis-inhibition experiments agree with the involvement of a carrier-mediated transport in addition to passive diffusion. The decrease of passive uptake resulting from resveratrol binding to serum proteins brings to light a mediated mechanism in physiological situation.

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Materials and methods

Chemicals. Trans-resveratrol was purchased from Sigma (St. Louis, MO, USA). [3H]-trans-resveratrol (specific activity: 74 GBq/mmol) labelled in ortho and para of benzenic rings was prepared for us by Amersham. Stock solutions of trans-resveratrol were prepared in absolute ethanol or in dimethyl sulfoxide (DMSO), wrapped in aluminium foil for protection against light, and stored at 4 °C when not in use. Resveratrol 3,5,4-triacetate and ε-viniferin were a gift from Actichem (Montauban, Fr). Bovine

Fluorescence measurement of resveratrol uptake

Slides were prepared from HepG2 cells preincubated with resveratrol during various times and then fixed. They were observed by fluorescence microscopy (Fig. 1). Resveratrol intrinsic fluorescence allowed a direct visualization of its intracellular uptake. A green fluorescence was observed in treated cells, markedly higher than basal fluorescence of untreated cells. Green fluorescence was obvious after incubation times less than 2 min. Fluorescence appeared to be distributed among the whole cell.

Discussion

For the first time, we report that the fluorescent properties of resveratrol allowed an easy method to follow the transport of this polyphenol by fluorescence microscopy. Such a method is also convenient for the study of the transport of resveratrol derivatives such as resveratrol triacetate and ε-viniferin (data not shown).

The observation of fluorescent slides showed that resveratrol is present essentially in cytoplasm. The nucleolar localization of resveratrol remains to be explained. A

Acknowledgements

This study was supported by the “Conseil Régional de Bourgogne,” BIVB, IREB, ONIVINS, and the “Ligue Bourguignonne contre le Cancer.” Thanks to Mr. O. Bocrie for preliminary experiments.

References (27)

Cited by (0)

1

The first two authors have equally contributed to this work.

2

Present address: Pharmakokinetic and Metabolism, Sanofi Synthélabo Recherche, 91380 Chilly-Marzarin, France.

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