Retinoic acid synergizes ATO-mediated cytotoxicity by precluding Nrf2 activity in AML cells.

BACKGROUND: Standard therapy for acute promyelocytic leukaemia (APL) includes retinoic acid (all-trans retinoic acid (ATRA)), which promotes differentiation of promyelocytic blasts. Although co-administration of arsenic trioxide (ATO) with ATRA has emerged as an effective option to treat APL, the molecular basis of this effect remains unclear. METHODS: Four leukaemia cancer human models (HL60, THP-1, NBR4 and NBR4-R2 cells) were treated either with ATO alone or ATO plus ATRA. Cancer cell survival was monitored by trypan blue exclusion and DEVDase activity assays. Gene and protein expression changes were assessed by RT-PCR and western blot. RESULTS: ATO induced an antioxidant response characterised by Nrf2 nuclear translocation and enhanced transcription of downstream target genes (that is, HO-1, NQO1, GCLM, ferritin). In cells exposed to ATO plus ATRA, the Nrf2 nuclear translocation was prevented and cytotoxicity was enhanced. HO-1 overexpression reversed partially the cytotoxicity by ATRA-ATO in HL60 cells. The inhibitory effects of ATRA on ATO-mediated responses were not observed in either the ATRA-resistant NB4-R2 cells or in NB4 cells pre-incubated with the RARα antagonist Ro-41-52-53. CONCLUSIONS: The augmented cytotoxicity observed in leukaemia cells following combined ATO-ATRA treatment is likely due to inhibition of Nrf2 activity, thus explaining the efficacy of combined ATO-ATRA treatment in the APL therapy.

Antiproliferative effects of phenylaminonaphthoquinones are increased by ascorbate and associated with the appearance of a senescent phenotype in human bladder cancer cells.

Quinone-containing molecules have been developed against cancer mainly for their redox cycling ability leading to reactive oxygen species (ROS) formation. We have previously shown that donor-acceptor phenylaminonaphthoquinones are biologically active against a panel of cancer cells. In this report, we explored the mechanisms involved in cancer cell growth inhibition caused by two phenylaminonaphthoquinones, namely Q7 and Q9, with or without ascorbate (ASC). The results show that Q7 and Q9 are both redox cyclers able to form ROS, which strongly inhibit the proliferation of T24 cells. Q9 was a better redox cycler than Q7 because of marked stabilization of the semiquinone radical species arising from its reduction by ascorbate. Indeed, ASC dramatically enhances the inhibitory effect of Q9 on cell proliferation. Q9 plus ASC impairs the cell cycle, causing a decrease in the number of cells in the G2/M phase without involving other cell cycle regulating key proteins. Moreover, Q9 plus ASC influences the MAPK signaling pathways, provoking the appearance of a senescent cancer cell phenotype and ultimately leading to necrotic-like cell death. Because cellular senescence limits the replicative capacity of cells, our results suggest that induction of senescence may be exploited as a basis for new approaches to cancer therapy.

Gut microbiota-derived propionate reduces cancer cell proliferation in the liver.

BACKGROUND: Metabolites released by the gut microbiota may influence host metabolism and immunity. We have tested the hypothesis that inulin-type fructans (ITF), by promoting microbial production of short-chain fatty acids (SCFA), influence cancer cell proliferation outside the gut. METHODS: Mice transplanted with Bcr-Abl-transfected BaF3 cells, received ITF in their drinking water. Gut microbiota was analysed by 16S rDNA polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and qPCR. Serum Short-chain fatty acids were quantified by UHPLC-MS. Cell proliferation was evaluated in vivo, by molecular biology and histology, and in vitro. RESULTS: Inulin-type fructans treatment reduces hepatic BaF3 cell infiltration, lessens inflammation and increases portal propionate concentration. In vitro, propionate reduces BaF3 cell growth through a cAMP level-dependent pathway. Furthermore, the activation of free fatty acid receptor 2 (FFA2), a Gi/Gq-protein-coupled receptor also known as GPR43 and that binds propionate, lessens the proliferation of BaF3 and other human cancer cell lines. CONCLUSION: We show for the first time that the fermentation of nutrients such as ITF into propionate can counteract malignant cell proliferation in the liver tissue. Our results support the interest of FFA2 activation as a new strategy for cancer therapeutics. This study highlights the importance of research focusing on gut microbes-host interactions for managing systemic and severe diseases such as leukaemia.

Inhibition of cell proliferation and migration by oxidative stress from ascorbate-driven juglone redox cycling in human bladder-derived T24 cells.

The effects of juglone on T24 cells were assessed in the presence and absence of ascorbate. The EC(50) value for juglone at 24 h decreased from 28.5 µM to 6.3 µM in the presence of ascorbate. In juglone-treated cells, ascorbate increased ROS formation (4-fold) and depleted GSH (65%). N-acetylcysteine or catalase restricted the juglone/ascorbate-mediated effects, highlighting the role of oxidative stress in juglone cytotoxicity. Juglone alone or associated with ascorbate did not cause caspase-3 activation or PARP cleavage, suggesting necrosis-like cell death. DNA damage and the mild ER stress caused by juglone were both enhanced by ascorbate. In cells treated with juglone (1-5 µM), a concentration-dependent decrease in cell proliferation was observed. Ascorbate did not impair cell proliferation but its association with juglone led to a clonogenic death state. The motility of ascorbate-treated cells was not affected. Juglone slightly restricted motility, but cells lost their ability to migrate most noticeably when treated with juglone plus ascorbate. We postulate that juglone kills cells by a necrosis-like mechanism inhibiting cell proliferation and the motility of T24 cells. These effects are enhanced in the presence of ascorbate.

Targeting cancer cells by an oxidant-based therapy.

Despite the progress achieved in chemo- and radiotherapy, cancer is still a leading life-threatening pathology. In that sense, there is a need for novel therapeutic strategies based on our current knowledge of cancer biology. Among the phenotypical features of cancer cells, two of them are of particular interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress, both resulting from the combination of high anabolic needs and hypoxic growth conditions. By using menadione (vitamin K3) and ascorbate (vitamin C), we took advantage of these features to develop an original approach that consists in the exposure of cancer cells to an oxidant insult. When used in combination, these compounds exhibit a synergistic action and are devoid of major toxicity in vivo. Thus, this review is dedicated to the analysis of the molecular pathways by which this promising combination exerts its antitumoural effect.

Role of ATP and glycogen reserves in both paracetamol sulfation and glucuronidation by cultured precision-cut rat liver slices.

Precision-cut rat liver slices (PCLS) were used to investigate the formation of paracetamol conjugates. The time course of biochemical markers such as ATP and GSH content, glycogen levels and protein synthesis rates was recorded over a period of time of 26 h and taken as index of slices viability. Low values of ATP (3.6 nmol/mg prot), GSH (7.1 nmol/mg prot) and protein synthesis rates (94.1 pmol leu/mg prot x min(-1)) were initially observed. Thereafter, they gradually recovered up to 6 h but decreased values were seen after 20 h. Glycogen, however, dropped rapidly during the first 6 h, being no longer detected after 20 h of incubation. The reincubation of PCLS in a fresh medium for 6 h allowed a strong recovery of GSH, ATP and protein synthesis rates, but no gluconeogenesis was observed. Meanwhile, paracetamol sulfate formation was fairly constant (about 3 microg/mg protein) while glucuronide gradually disappeared. The amount of both UGT1A1 and ST1A1 did not correlate with their respective enzymatic activities. We suggest that loss of glycogen impair glucuronide conjugation by decreasing the availability of UDPGA, and that low values of ATP are largely enough to support sulfotransferase activity.

Effect of cold hypoxic storage and reoxygenation at 37 degrees C of cultured precision-cut rat liver slices on paracetamol metabolism.

The influence of cold storage and reoxygenation at 37 degrees C of precision-cut rat liver slices on paracetamol metabolism was studied. A depressed metabolism was observed after cold preservation, but during reoxygenation at 37 degrees C slices were capable of synthesizing proteins and maintaining both glutathione and ATP levels. Recovery was faster in slices under aerobic cold conditions than in those under cold hypoxia. Glycogen levels were dramatically decreased under both conditions and subsequent reoxygenation at 37 degrees C still increased the glycogenolysis. Xenobiotic metabolism after reoxygenation of cold-preserved slices shows that glucuronide and sulfate conjugates of paracetamol represent about 50% of those of fresh slices at zero time. The amounts of phase II apoproteins were virtually unchanged, thus suggesting that loss of enzyme activities are most probably due to lack of cofactors. Reoxygenation at 37 degrees C did not impair cell metabolism, and a potential role for nitric oxide and other cytokines released form Kupffer cells appears unlikely since nitrite was not formed after bacterial endotoxin stimulation. The maintenance of energetic stores during cold preservation appears, therefore, to be a critical issue for the survival and metabolic activity of cells.

Activation of Poly(ADP-Ribose)Polymerase in rat hepatocytes does not contribute to their cell death by oxidative stress.

Oxidative stress induced by tert-butyl hydroperoxide (tBOOH) in freshly isolated rat hepatocytes caused DNA damage and loss of membrane integrity. Such DNA lesions are likely to be single strand breaks since neither caryolysis nor chromatine condensation was seen in electron micrographs from tBOOH-treated cells. In addition, pulsed field gel electrophoresis of genomic DNA from both control and tBOOH-treated hepatocytes showed similar profiles, indicating the absence of internucleosomal DNA cleavage, a classical reflection of apoptotic endonuclease activity. The activation of the repair enzyme poly(ADP-ribose)polymerase (PARP) following DNA damage by tBOOH induced a dramatic drop in both NAD(+) and ATP. The inhibition of PARP by 3-aminobenzamide enhanced DNA damage by tBOOH, restored NAD(+) and ATP levels, but did not result in better survival against cell killing by tBOOH. The lack of the protective effect of PARP inhibitor, therefore, does not implicate PARP in the mechanism of tBOOH-induced cytotoxicity. Electron micrographs also show no mitochondrial swelling in cells under oxidative stress, but such organelles were mainly located around the nucleus, a picture already observed in autoschizis, a new suggested kind of cell death which shows both apoptotic and necrotic morphological characteristics.

Protein S-thiolation can mediate the inhibition of protein synthesis induced by tert-butyl hydroperoxide in isolated rat hepatocytes.

A rapid inhibition of protein synthesis is observed when isolated rat hepatocytes are incubated in the presence of 0.25-0.5 mM of tert-butyl hydroperoxide (tBOOH). Such an inhibition occurs in the absence of a cytolytic effect by tBOOH. Iron chelators (o-phenanthroline and desferrioxiamine), protected against oxidative cell death, but they did not modify the inhibition of protein synthesis caused by tBOOH (0.5 mM), suggesting that free radicals are less implicated in such an impairment. Electron micrographs of hepatocytes under oxidative stress show disaggregation of polyribosomes but not oxidative alterations, such as blebs or mitochondrial swelling. Protein synthesis inhibition is accompanied by a decrease in reduced glutathione (GSH) and an increase in glutathione disulfide (GSSG) and the level of protein S-thiolation (protein mixed disulfides formation). Such an increase of GSSG appears as a critical event since diethylmaleate (DEM) at 0.2 mM reduced GSH content by more than 50% but did not affect either GSSG content or protein synthesis. The addition of exogenous GSH and N-acetylcysteine (NAC) to tBOOH-treated hepatocytes significantly reduced the formation of protein mixed disulfides and restored the depressed protein synthesis either completely or partially. We suggest that S-thiolation of some key proteins may be involved in protein synthesis inhibition by tBOOH.

Hypoxia increases the association of 4E-binding protein 1 with the initiation factor 4E in isolated rat hepatocytes.

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.

Thermic transition and glycolytic capacity as critical events in the survival of rat liver slices after overnight cold hypoxic preservation.

Cellular survival and hypoxia-reoxygenation injury in overnight cold-preserved liver slices (+/-20 h at 4 degrees C) were investigated. Increased cell death after overnight cold hypoxia depended more on temperature than on the reoxygenation process itself. Fructose (at 50 mM) added before the onset of hypoxia improved survival at the end of 20 h of cold hypoxia over Krebs- or glucose-treated slices. Such a protective effect by fructose was also seen during the normothermic (37 degrees C) reoxygenation of previously cold hypoxic-preserved slices, but only in the absence and not in the presence of tert-butyl hydroperoxide, a model compound widely used to induce an oxidative stress. The protection by fructose was equivalent to that observed when liver slices were incubated in the University of Wisconsin solution (UW). Finally, the morphological study of haematoxylin and eosin (H & E)-stained slices has shown cytoplasmic vacuoles during the reoxygenation step, which were more pronounced in UW-treated than in fructose-treated slices.

Role of protein-phosphorylation events in the anoxia signal-transduction pathway leading to the inhibition of total protein synthesis in isolated hepatocytes.

Incubation of isolated hepatocytes under N2/CO2 (no O2) 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-kinase 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 Na3VO4 (1 mM) reduced the incorporation of [14C]Leu into proteins of hepatocytes maintained under aerobic conditions (93.3 kPa O2). 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 O2. 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.

Protective effect of fructose on survival and metabolic capacities of hepatocytes kept overnight under cold hypoxia before normothermic reoxygenation.

The protective effect of fructose with regard to hypoxia-induced cell injury in overnight cold preserved hepatocytes (20 hr at 4 degrees C) was investigated. The addition of fructose (at 10 and 20 mM) resulted in an improved survival of hepatocytes during their normothermic (37 degrees C) reoxygenation, irrespective of the time of fructose addition before the onset of hypoxia (i.e. 10, 20 or 30 min). Such a protective effect was even higher than that observed when hepatocytes were incubated in the University of Wisconsin solution (UW). Moreover, neither Desferal (an iron chelator) nor adenosine (an ATP precursor), nor other carbohydrates (glucose, galactose and the antioxidant mannitol) were able to protect cells against such an hypoxia-mediated injury. The intracellular ATP content was lower in both adenosine- and fructose-treated hepatocytes than in control untreated cells. However, the cellular metabolic capacities such as protein synthesis and gluconeogenesis from lactate recovered faster during reoxygenation of previously hypoxic fructose-treated cells compared with both control and adenosine-treated cells.

Adenosine stimulates calcium influx in isolated rat hepatocytes.

The mechanism of stimulation of Ca2+ entry into hepatocytes by adenosine was investigated. When Fura-2-loaded hepatocytes were suspended in a nominally Ca(2+)-free buffer, adenosine produced only a small transient increase in the cytosolic free Ca2+ concentration ([Ca2+)i). However, on restoration of an extracellular Ca2+ concentration of 1.3 mM, a rapid increase in [Ca2+]i occurred, which indicates activation of a Ca(2+)-influx pathway. Adenosine augmented the rate of Ca2+ influx triggered by maximally effective concentrations of thapsigargin or cAMP, but was without effect on the rate of Ca2+ entry that resulted from phospholipase-C-linked-receptor activation by maximally effective concentrations of vasopressin or ATP. However, in contrast to vasopression and ATP, adenosine did not stimulate Mn2+ entry. The rate of Mn2+ influx after stimulation of the hepatocytes with vasopressin was not increased by adenosine treatment. The stimulation of hepatocytes with adenosine did not result in significant accumulation of inositol phosphates or cAMP. Furthermore, the rate of adenosine-induced Ca2+ entry in hepatocytes was only slightly reduced in the presence of the P1 purinoceptor antagonist 8-phenyltheophylline. In contrast, the receptor-mediated-Ca(2+)-entry antagonist SK&F 96365 nearly completely blocked the Ca(2+)-entry response without any effect on internal-Ca(2+)-pool mobilisation by adenosine. It is concluded that adenosine activates the internal-pool-regulated pathway of Ca2+ entry and an additional pathway that appears comparable to the previously reported receptor-dependent pathway, except that Mn2+ entry is not stimulated.

Targeting of drug to the hepatocytes by fatty acids. Influence of the carrier (albumin or galactosylated albumin) on the fate of the fatty acids and their analogs.

PURPOSE: The aim of this study was to evaluate the potential of fatty acids as shuttles to deliver xenobiotic inside the hepatocytes as well as to study the mechanism of incorporation into isolated hepatocytes when bound to native albumin or galactosylated albumin. Theoretically, they can enter into the hepatocytes after recognition of the Fatty Acid Binding Protein (FABPPM), or remain bound to galactosylated proteins and enter into these cells by a process known as receptor mediated endocytosis after selective recognition of the asialoglycoprotein receptor (ASGPR). METHODS: We synthesized a 3H-benzoyl adduct of lauric acid (BLA) (benzoyl adduct chosen to mimic any low molecular weight drug or contrast agent), and compared the behavior of BLA with oleic acid for their binding properties to carrier-proteins and the uptake mechanism by isolated hepatocytes. RESULTS: No significant difference was found in the binding properties of BLA for albumin and galactosylated albumin. The incorporation into the hepatocytes was found essentially depending on the FABPPM transport system whenever BLA was bound to albumin or to galactosylated albumin in the incubation medium: indeed, the transport was inhibited by phloretin (inhibitor of sodium dependent transport), increased when the free part of BLA was higher, and BLA was recovered in the cytosolic fraction of the hepatocytes. CONCLUSIONS: This study showed the convenience in using fatty acids as drug carriers possessing tropism for the hepatocytes.

Oxidative DNA damage by t-butyl hydroperoxide causes DNA single strand breaks which is not linked to cell lysis. A mechanistic study in freshly isolated rat hepatocytes.

In rat hepatocytes, 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 interconnected. Since o-phenanthroline prevents against tBOOH-mediated effects on both DNA and membrane integrity, we discussed about a putative role of iron.

Homocysteine enhances the inhibitory effect of extracellular adenosine on the synthesis of proteins in isolated rat hepatocytes.

Previous work has shown that extracellular adenosine inhibits the incorporation of radiolabelled leucine into proteins in isolated rat hepatocytes [Tinton, Lefebvre, Cousin and Buc Calderon (1993) Biochim. Biophys. Acta 1176, 1-6]. In this study, we investigated whether its metabolism into adenine nucleotides, inosine or S-adenosylhomocysteine (AdoHcy) is required to induce such an impairment. Incubation of isolated hepatocytes in the presence of adenosine at 0.5 or 1 mM reduces the synthesis of proteins by about 45% after 120 min of incubation. Such an inhibition occurred without cell lysis and was not modified by adding the adenosine kinase inhibitor 5-iodotubercidin (15 microM) or the adenosine deaminase inhibitor coformycin (0.1 microM). It is therefore unlikely that the anabolic and catabolic pathways of adenosine are involved in the inhibition of protein synthesis. Adenosine (1 mM) increased the level of AdoHcy and S-adenosylmethionine by 20- and 5-fold respectively after 60 min of incubation and reduced the methylation index. These events as well as the inhibition of protein synthesis were strongly enhanced in the presence of L-homocysteine (2 mM). It is therefore concluded that the metabolism of adenosine into AdoHcy, which is known to be a potent inhibitor of cellular methylation reactions, may play an important role in the control of translation.

Inhibition of protein synthesis induced by adenine nucleotides requires their metabolism into adenosine.

Adenine nucleotides and adenosine inhibit the incorporation of radiolabelled leucine into proteins of isolated hepatocytes. Impairment occurred with nucleotides which can be converted into 9-beta-D-ribofuranosyladenine (adenosine) but was not observed after treatment with adenine or AMPCPP (the alpha, beta-methylene analogue of ATP). Metabolism into adenosine was further suggested by the increase in cellular ATP levels following treatment of hepatocytes with ATP, adenosine or AMPPCP (the beta, gamma-methylene ATP analogue) while AMPCPP was without any significant effect. The inhibition of protein synthesis caused by adenosine was not due to a lytic effect nor to a general disturbance in hepatic functions and was reversed when the cells were washed and transferred to a nucleoside-free medium. This impairment, however, was not coupled to the activation of adenylate cyclase, as preincubation of hepatocytes with P1 purinoceptor antagonists failed to prevent protein synthesis inhibition. In contrast, L-homocysteine enhanced the inhibitory effect of adenosine on the incorporation of radiolabelled leucine into proteins. Our results thus suggest that the inhibition of protein synthesis caused by adenine nucleotides requires their conversion into adenosine. They also indicate that the inhibitory effect of adenosine does not involve a receptor-mediated effect but may be related to an increase in S-adenosylhomocysteine content and a subsequent low level of macromolecule methylation.

Adenosine inhibits protein synthesis in isolated rat hepatocytes. Evidence for a lack of involvement of intracellular calcium in the mechanism of inhibition.

Extracellularly added adenosine and ATP are potent inhibitors of protein synthesis in liver cells. In this study, the possible involvement of Ca2+ in the mechanism of inhibition of protein synthesis by adenosine was investigated. Stimulation of freshly isolated hepatocytes with adenosine or ATP, at concentrations that impaired protein synthesis, induced an increase in the cytosolic free Ca2+ concentration ([Ca2+]i). However, there was no correlation between the increase in [Ca2+]i and inhibition of radiolabelled leucine incorporation into proteins. Thus, the stimulation of hepatocytes with the V1-receptor agonist, vasopressin, or with the nucleotide triphosphates, UTP and GTP, elicited changes in [Ca2+]i similar to those observed after ATP or adenosine addition, but did not affect protein synthesis. ATP produced near complete discharge of Ca2+ from the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool in isolated hepatocytes, whereas adenosine only had a partial effect. Depletion of the hormone-sensitive Ca2+ pool by adenosine was transient. In contrast, prolonged depletion of internal Ca2+ by thapsigargin resulted in the inhibition of protein synthesis in hepatocytes. However, the inhibition of radiolabelled leucine incorporation into proteins by thapsigargin was further augmented by the additional presence of adenosine. These results show that the inhibition of protein synthesis by adenosine in isolated hepatocytes is not mediated by an increase in [Ca2+]i or depletion of internal pool(s) sensitive to inositol 1,4,5-trisphosphate or thapsigargin.

Desferal prevents against cell lysis induced by hydrogen peroxide to hypoxic hepatocytes: a role for free iron in hypoxia-mediated cellular injury.

Isolated hepatocytes incubated under hypoxic conditions were more sensitive to H2O2-mediated injury as compared to cells kept under aerobic conditions, but only for the highest H2O2 concentration tested (8 mM). At lower concentrations (2 and 4 mM) cells were still able to detoxify H2O2 even under hypoxic conditions. Reoxygenation of hypoxic hepatocytes did not result in a cytolytic effect, whereas reoxygenation in the presence of H2O2 resulted in an enhanced cytotoxicity. The duration of previous hypoxia (before H2O2 addition) did not affect the lytic effect induced by H2O2. Enzymatic activities of both catalase and glutathione peroxidase were unchanged over 2 h of incubation under hypoxic conditions. Preincubation of hepatocytes in the presence of Desferal (5 mM) resulted in the abolition of H2O2-mediated lytic effects. A role for free iron, released from intracellular stores and acting on H2O2 to yield reactive oxygen species is discussed.