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Keratinocytes are potential targets of lipid peroxidation products (alpha,beta-unsaturated aldehydes) generated in the skin following UV exposure, among which the most abundant and toxic product is 4-hydroxy-trans-2,3-nonenal (HNE). The aim of this study was to investigate the ability of keratinocytes (NCTC2544 cell lines) to detoxify HNE, through characterization of metabolites, until now never demonstrated, using a combined analytical approach (liquid chromatography (LC) and liquid chromatography/mass spectrometry (LC/MS)). Incubation of cells with HNE (up to 200 micro M) was performed in order to evaluate the ability of the cells to detoxify this toxic aldehyde, and indicated that the cell viability was maintained under these conditions. LC analysis of the extracellular media from keratinocytes incubated with 100 micro M HNE shows a time-dependent decrease of HNE, disappearance from the medium within 2 h and concomitant formation of two unconjugated (phase I) metabolites, 4-hydroxy-2-nonenoic acid (HNA) and 1,4-dihydroxy-2-nonene (DHN), which were both identified and quantified by LC and accounted for 48.8 +/- 4.6% of the HNE dose. Four additional metabolites were identified in the extracellular medium by reversed-phase LC coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with positive and negative ion detection as Michael adducts (phase II metabolites), arising by the addition of the nucleophilic sulfur of glutathione (GSH) to the electrophilic C-3 of HNE, followed by oxidation-reduction enzymatic processes. The GSH-HNE conjugates were (a) S-(4-hydroxynonanal-3-yl)glutathione, (b) S-(1,4-dihydroxy-nonane-3-yl)glutathione, (c) S-(4-oxononanal-3-yl)glutathione and (d) S-(4-oxo-nonan-1-ol-3-yl)glutathione, and accounted for 52.3 +/- 5.8% of the HNE dose (35 nmol mg(-1) protein), as estimated indirectly by measuring the extent of cellular GSH consumption (18.7 +/- 1.8 nmol mg(-1) protein). The time course of HNE biotransformation was then determined by monitoring the formation of metabolites inside and outside the cell at different times after HNE addition (5-120 min). A time-dependent and almost linear formation inside the cell was observed for all the metabolites (plateau after 15 min of incubation), followed by a rapid decay and a concomitant increase in the extracellular medium (plateau of formation after 60 min). This confirms that HNE diffuses into the cell where is totally metabolized through phase I and phase II reactions to unreactive products, which are then exported outside the cell. This is the first demonstration that skin epidermal cells are able to detoxify the cytotoxic products of lipid peroxidation.

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N6‐isopentenyladenosine (i6A), a member of the cytokinin family of plant hormones, has potent in vitro antitumour activity in different types of human epithelial cancer cell lines. Gene expression profile analysis of i6A‐treated cells revealed induction of genes (e.g., PPP1R15A, DNAJB9, DDIT3, and HBP1) involved in the negative regulation of cell cycle progression and reportedly up‐regulated during cell cycle arrest in stress conditions. Of 6 i6A analogues synthesized, only the 1 with a saturated double bond of the isopentenyl side chain had in vitro antitumour activity, although weaker than that of i6A, suggesting that i6A biological activity is highly linked to its structure. In vivo analysis of i6A and the active analogue revealed no significant inhibition of cancer cell growth in mice by either reagent. Thus, although i6A may inhibit cell proliferation by regulating the cell cycle, further studies are needed to identify active analogues potentially useful in vivo. © 2008 Wiley‐Liss, Inc.

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