The potential use of stem/progenitor cells as alternative cell sources to mature hepatocytes remains basically dependent on their ability to exhibit some, if not all, the metabolic liver functions. In the current study, four major liver functions were investigated in adult derived human liver stem/progenitor cell (ADHLSCs) populations submitted to in vitro hepatogenic differentiation: gluconeogenesis, ammonia detoxification, and activity of phase I and phase II drug-metabolizing enzymes. These acquired hepatic activities were compared to those of primary adult human hepatocytes, the standard reference. Amino acid content was also investigated after hepatogenic differentiation. Differentiated ADHLSCs display higher de novo synthesis of glucose correlated to an increased activity of glucose-6 phosphatase and mRNA expression of key related enzymes. Differentiated ADHLSCs are also able to metabolize ammonium chloride and to produce urea. This was correlated to an increase in the mRNA expression of relevant key enzymes such arginase. With respect to drug metabolism, differentiated ADHLSCs express mRNAs of all the major cytochromes investigated, among which the CYP3A4 isoform (the most important drug-metabolizing enzyme). Such increased expression is correlated to an enhanced phase I activity as independently demonstrated using fluorescence-based assays. Phase II enzyme activity and amino acid levels also show a significant enhancement in differentiated ADHLSCs. The current study, according to data independently obtained in different labs, demonstrates that in vitro differentiated ADHLSCs are able to display advanced liver metabolic functions supporting the possibility to develop them as potential alternatives to primary hepatocytes for in vitro settings.

Incubation of hepatocytes under hypoxia increases binding of translation initiation factor eIF-4E to its inhibitory regulator 4E-BP1, and this correlates with dephosphorylation of 4E-BP1. Rapamycin induced the same effect in aerobic cells but no additive effect was observed when hypoxic cells were treated with rapamycin. This enhanced association of 4E-BP1 with eIF-4E might be mediated by mTOR. Nevertheless, only hypoxia produces a rapid inhibition of protein synthesis. Although hypoxia might be signalling via the rapamycin-sensitive pathway by changing eIF-4E availability, such a pathway is unlikely to be responsible for the depression in overall protein synthesis under hypoxia.z 1999 Federation of European Biochemical Societies.

In rat hepatoeytes, DNA damage by t-butyl hydroperoxide (tBOOH) was measured by using the fluorimetric analysis of alkaline DNA unwinding. The electrophoretic profile of genomic DNA suggests single rather than double DNA strand breaks formation. Oxidative DNA modifications, measured as increased 8-hydroxy-deoxyguanosine content, were not detected. Lysis of hepatocytes and DNA strand breaks induced by tBOOH did not correlate, indicating that both processes are not interconm~ted. Since o-phenantbroline prevents against tBOOH-mediated effects on both DNA and membrane integrity, we discussed about a putative role of iron.I~ ey words: DNA fragmentation; Oxidative stress; Organic hydroperoxide; Free radical; Rat hepatocyte This work was undertaken with the aim to study and characterize the DNA damage induced by tBOOH. We further analysed whether this putative DNA damage was linked to the tBOOH-mediated cell death. Oxidant injury was evaluated by following the formation of DNA strand breaks, the time course of LDH leakage and the formation of the DNA adduct 8-hydroxy-deoxyguanosine (8-OH-dG). The extent and the nature of DNA strand breakage were evaluated by the method of fluorometric analysis of the rate of alkaline DNA unwinding (FADU) and by the electrophoretic profile of genomic DNA run on agarose gel, respectively. The cytotoxicity of tBOOH was modulated by using the inhibitor of lipid peroxidation N,N'-diphenyl-p-phenylenediamine (DPPD), the iron chelator o-phenanthroline (oPT) and zinc sulfate and aurintricarboxylic acid (ATCA) as an endonuclease inhibitor.

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Incubation of isolated hepatocytes under N,/COZ (no 0,) produced a rapid and strong inhibition of overall polypeptide biosynthesis, which was neither related to cell death nor to the appearance of specific stress proteins. Treatment of the cells with the tyrosine-kinase inhibitor genistein or with the serine/ threonine-protein-kinaqe inhibitor H7 did not modify the impairment of protein synthesis induced by oxygen deprivation, indicating that such signal-transduction pathways are probably not involved in the anoxia-mediated effect. Okadaic acid (100 nM) and Na,VO, (1 mM) reduced the incorporation of[I4C]Leu into proteins of hepatocytes maintained under aerobic conditions (93.3 kPa OJ. The effects of oxygen deprivation and okadaic acid were additive, whereas sodium vanadate did not enhance the impairment of protein synthesis induced by anoxia. This observation suggests that a common mechanism, involving the net phosphorylation of protein tyrosine residues, that is insensitive to genistein might participate in the negative control of the translation induced by oxygen deprivation. The effect of anoxia on the synthesis of proteins was fully and rapidly reversible upon the restoration of oxygen supply, thus indicating that hepatocytes are able to sense 0,. Although high concentrations of cobalt chloride partially mimic the effect of oxygen deprivation on protein biosynthesis, the nature of such an oxygen sensor remains unknown, and appears unlikely to be a part of a classic haem protein.Keywords: anoxia; protein synthesis ; protein phosphorylation ; isolated hepatocytes.Hypoxia produces a variety of physiological and biochemical adaptations so that energy can be supplied to the tissues to maintain cellular activities [l -41. A decrease in the production of almost all polypeptides appears to be an important feature of oxygen deprivation in numerous cell types [5-71. Thus, protein synthesis, a major function of the liver, is inhibited as soon as hepatocytes are kept under hypoxic conditions, indicating that cells react to a change in PO, levels rather than to a change in either ATP content or ATP/ADP ratio [8, 91. Moreover, such an inhibition was not related to a decreased amino acid uptake or to enhanced proteolytic activities, but also occurred in cold-preserved hepatocytes [lo] and in acute ischemic rat liver [ll].In mammalian cells, reduced 0, tension modulates genes for proteins such as erythropoietin [ 12, 131, glycolytic enzymes, which increase anaerobic ATP synthesis [14-161, vascular endothelial growth factor [16, 171, and transcription factors, such as hypoxia inducible factor-I [IS], nuclear factor KB and activator protein 1 [19]. Such responses indicate that cells are able to sense oxygen, but the nature of the oxygen sensor and the mechanisms involved in the hypoxia signal-transduction pathway are not fully understood.Correspondence to P.