Modulation of ethanol effect on hepatocyte proliferation by polyamines.

An occurrence and a magnitude of alcoholic liver diseases depend on the balance between ethanol-induced injury and liver regeneration. Like ethanol, polyamines including putrescine, spermidine, and spermine modulate cell proliferation. Thus, the purpose of this study was to evaluate the relationship between effect of ethanol on hepatocyte (HC) proliferation and polyamine metabolism using the HepaRG cell model. Results showed that ethanol effect in proliferating HepaRG cells was associated with a decrease in intracellular polyamine levels and ornithine decarboxylase (ODC) activity. Ethanol also induced disorders in expression of genes coding for polyamine-metabolizing enzymes. The α-difluoromethyl ornithine, an irreversible inhibitor of ODC, amplified ethanol toxicity on cell viability, protein level, and DNA synthesis through accentuation of polyamine depletion in proliferating HepaRG cells. Conversely, putrescine reversed ethanol effect on cell proliferation parameters. In conclusion, this study suggested that ethanol effect on HC proliferation was closely related to polyamine metabolism and that manipulation of this metabolism by putrescine could protect against the anti-proliferative activity of ethanol.

Antiproliferative and apoptotic effects in rat and human hepatoma cell cultures of the orally active iron chelator ICL670 compared to CP20: a possible relationship with polyamine metabolism.

OBJECTIVE: Iron loading has been observed to have a hyperproliferative effect on hepatocytes in vitro and on tumour cells in vivo; removal of this iron being required to induce antitumour activity. MATERIAL AND METHODS: Antiproliferative effects of orally active tridentate iron chelator ICL670 (deferasirox) and bidentate iron chelator CP20 (deferiprone), mediated through the chelation of intracellular iron, were compared in rat hepatoma cell line FAO and human hepatoma cell line HUH7. RESULTS: In FAO cell cultures, we have shown that ICL670 decreased cell viability and DNA replication and induced apoptosis more efficiently than an iron-binding equivalent concentration of CP20. Moreover, ICL670 decreased significantly the number of the cells in G(2)-M phase. In the HUH7 cell cultures, ICL670 and a four-time higher iron-binding equivalent concentration of CP20, decreased cell viability and DNA replication in the same range. CP20 increased the number of the cells in G(2)-M phase. However, ICL670 inhibited polyamine biosynthesis by decreasing ornithine decarboxylase mRNA level; in contrast, CP20 increased polyamine biosynthesis, particularly putrescine level, by stimulating spermidine-spermine N(1)-acetyl transferase activity that could activate the polyamine retro-conversion pathway. By mass spectrometry, we observed that ICL670 cellular uptake was six times higher than CP20. CONCLUSIONS: These results suggest that ICL670 has a powerful antitumoural effect and blocks cell proliferation in neoplastic cells by a pathway different from that of CP20 and may constitute a potential adjuvant drug for anticancer therapy.

[HFE hemochromatosis: pathogenic and diagnostic approach]. / Hémochromatose HFE: approche pathogénique et diagnostique.

HFE hemochromatosis is the most frequent genetic iron overload disease. It is linked to the C282Y mutation of the HFE protein, protein encoded by the HFE gene, which is located on chromosome 6. The mechanisms accounting for iron excess are not only digestive hyperabsorption of iron but also excessive recycling of macrophagic iron coming from erythrophagocytosis and secreted into the blood. Both mechanisms are linked to an HFE-related hepatic failure in producing hepcidin, a key hormone of body iron regulation. The marked phenotypic variability of C282Y homozygosity expression is likely related to both genetic and environmental factors. The HFE gene discovery has rendered non invasive the positive diagnostic of HFE hemochromatosis, which is now based first on an increased level of plasma transferrin saturation leading to the request of the HFE mutation. Then, hepatic MRI is a reliable method to quantify iron overload. The HFE gene discovery has also paved the road of an enlarged field of differential diagnoses corresponding to novel entities of non-HFE related genetic iron overload syndromes.

Modulation of alpha-fetoprotein, albumin and transferrin gene expression by cellular interactions and dexamethasone in cocultures of fetal rat hepatocytes.

Fetal hepatocytes were cultured alone or in association with primitive biliary cells (RLEC) in the presence or absence of dexamethasone. Cell-cell contacts were established 3 h or five days after hepatocyte seeding and their effects on hepatocyte growth and functional activities were evaluated in the presence or absence of dexamethasone. Establishment of cellular interactions with RLEC in coculture decreased hepatocyte growth, while it stimulated production of alpha-fetoprotein, albumin and transferrin. Addition of dexamethasone to coculture inhibited alpha-fetoprotein secretion and maintained the synthesis rate of albumin and transferrin together with an additional inhibition of DNA synthesis. The levels of mRNAs corresponding to the three proteins were also measured. We observed that the levels of alpha-fetoprotein, albumin and transferrin secretion in cocultures maintained in the presence or absence of dexamethasone were well correlated with the relative amounts of their corresponding mRNAs. Consequently, it may be assumed that the primitive mechanism involved in the increased functional activity of fetal hepatocytes in coculture is of pretranslational origin. Furthermore, the present data provide evidence that heterotypic interactions and dexamethasone act as distinct modulators of growth and maturation of fetal rat hepatocytes.

Chronic iron overload inhibits protein secretion by adult rat hepatocytes maintained in long-term primary culture.

Short-term pure cultures and long-term cocultures of adult rat hepatocytes with rat liver epithelial cells, presumably derived from primitive biliary cells, were used to define in vitro models of iron overloaded hepatocytes in order to understand the molecular mechanism responsible for liver damage occurring in patients with hemochromatosis. In vitro iron overload was obtained by daily addition of ferric nitrilotriacetate to the culture medium. A concentration of 20 microM ferric salt induced hepatocyte iron overload with minimal cytotoxicity as evaluated by cell viability, morphological changes of treated cells and cytosolic enzyme leakage into the culture medium. The effects of iron overload on protein biosynthesis and secretion were studied in both short-term pure cultures and long-term cocultures of hepatocytes. The amounts of intracellular and newly synthesized proteins were never modified by the iron treatment. Furthermore, neither the relative amounts of transferrin and albumin mRNAs nor their translational products were altered by iron overload. Moreover, no change in the transferrin isomeric forms were observed in treated cells. In contrast, a prolonged exposure of cocultured hepatocytes to 20 microM ferric salt led to a significant decrease in the amount of proteins secreted in the medium. This decrease included the two major secreted proteins, namely albumin and transferrin, and probably all other secreted proteins. These results demonstrate that iron loading alters neither the total nor the liver specific protein synthesis activity of cultured hepatocytes. They suggest that chronic overload may impede the protein secretion process.

EPR study of antioxidant activity of the iron chelators pyoverdin and hydroxypyrid-4-one in iron-loaded hepatocyte culture: comparison with that of desferrioxamine.

Iron supplementation of hepatocyte culture induced the production of lipid-derived radicals as shown by spin-trapping with alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN). The EPR signal corresponding to POBN/lipid-derived radicals (aN = 15.6 G aH = 2.6 G) was concentration dependent on iron (Fe-NTA) added to the culture medium (50, 100, 200 microM). It was also incubation time dependent (0 to 24 h). The EPR signal could be used as a marker for iron-induced lipid peroxidation. The antioxidant activity of two iron chelators, pyoverdin (Pa) and hydroxypyrid-4-one derivative (CP20) was compared with that of desferrioxamine (DFO) on iron-loaded hepatocyte culture. These compounds (100 microM) were tested either in pretreatment or simultaneously with Fe-NTA (100 microM). In each procedure, the EPR signal obtained from the cells supplemented with iron was substantially reduced in the presence of either DFO or CP20 but not with Pa. Moreover, the DFO and CP20 but not Pa showed protective effect on the leakage of the intracellular enzyme lactate dehydrogenase into the culture medium. The present study described a specific spin-trapping technique in conjunction with EPR spectroscopy that is able to demonstrate the cytoprotective effect of iron chelators, as shown by the elimination of lipid-derived radicals in iron-loaded hepatocyte culture.

Antioxidant and free radical scavenging activities of the iron chelators pyoverdin and hydroxypyrid-4-ones in iron-loaded hepatocyte cultures: comparison of their mechanism of protection with that of desferrioxamine.

The protective effect on iron-supplemented hepatocyte cultures of three iron chelators, pyoverdin Pa and hydroxypyrid-4-one derivatives CP20 and CP22, was compared to that of the widely known desferrioxamine B (Desferal:DFO), on the basis of two criteria: (a) their effectiveness in inhibiting free malondialdehyde (MDA) production as an index of iron-induced lipid peroxidation; and (b) their ability to reduce intracellular enzyme leakage. In view of these two markers of iron toxicity, the protective effect of these chelators was classified as follows: DFO > CP20 > or = CP22 > Pa. The mechanism of cellular protection was elucidated by investigating both the iron-chelating activity and the free radical scavenging property of these agents. As concerns the iron chelation, DFO and Pa exerted the same rank order as for cytoprotection (DFO > Pa). The free radical scavenging property toward hydroxyl radical .OH and peroxyl radical ROO. was investigated in a cell-free experimental model. The two siderophores, DFO and Pa, appeared to have a lower antiradical activity toward .OH than hydroxypyrid-4-one CP22. This .OH scavenging activity was classified as follows: CP22 >> Pa > DFO. Moreover, the chelators exhibited for the quenching of ROO. the same order of effectiveness as that observed for cellular protection: DFO > CP20 > or = CP22 > Pa. These data indicate that, in addition to the iron-chelating activity which represents the most important property for determining the protection capacity of these iron chelators, their free radical scavenging ability also must be taken into account. This direct demonstration of a strong association between the free radical scavenging activity and the protective effect of iron chelators further increases the prospects for the development and clinical applications of new oral chelating drugs.

In vitro demonstration of synergy between radionuclide and chemotherapy.

UNLABELLED: Radionuclide therapy is currently used in the treatment of some malignancies, including hepatocellular carcinoma. The effects of external beam radiotherapy are improved by combining it with chemotherapy. The aim of this study was to determine whether such a synergistic effect could be demonstrated in vitro with internal radiation therapy. METHODS: HepG2 cells were cultured from Day 0 to Day 8 under the following conditions: exposure for 4 hr on Day 2 to increasing concentrations of 5-fluorouracil (5FU), doxorubicin or cisplatin (CDDP); exposure from Day 2 to Day 8 to increasing concentrations of 131-iodide; exposure on Day 2 to low-toxicity doses of drugs for 4 hr, followed by exposure to 131I at increasing concentrations; and exposure to increasing concentrations of 131I from Day 2 to Day 8, with exposure for 4 hr on Day 6 to the drugs. Cell toxicity was assessed by enzyme release (lactate dehydrogenase and aspartate aminotransferase) in the culture medium and on cell survival (protein and tetrazolium dye test). All cultures were run in triplicate. RESULTS: A dose- and time-dependent toxicity was demonstrated with doxorubicin and CDDP but not with 5FU. When HepG2 cells were exposed to 131I, the toxicity was rather low, but significant, and was time- and dose-dependent. Treating these cells with combination radiotherapy and chemotherapy resulted in a toxicity that was significantly greater than that with 131I or chemotherapy drugs alone. CONCLUSION: The radiosensitivity of HepG2 cells is low; combining a chemotherapeutic drug with a radiotherapeutic agent improves the radiosensitivity in a synergistic fashion. This combination is thus able to strengthen the therapeutic effect of internal radiation therapy in different malignancies, particularly in hepatocellular carcinoma.

Influence of adrenalectomy on maturation of gonadotrophin function in the male rat.

The effects were studied of adrenalectomy performed at 25 days of age on the maturation of LH function and the testes during puberty (i.e. 30-50 days of age) in the male rat. In intact rats the plasma LH level increased and then decreased and the plasma testosterone level increased progressively. In adrenalectomized rats the plasma levels of LH and testosterone did not vary significantly over the same period of time. Adrenalectomy decreased the plasma levels of LH and testosterone. The different perturbations in hormonal balance induced a decrease in the size of the seminiferous tubules and a delay in spermatogenesis.

Inhibition of iron overload toxicity in rat hepatocyte cultures by pyoverdin Pf, the siderophore of Pseudomonas fluorescens.

The effect of the pyoverdin Pf (an iron chelating agent isolated and purified from Pseudomonas fluorescens CCM 2798) was studied on iron overloaded rat hepatocyte cultures. Iron overload was obtained by addition of 5-80 microM ferric nitrilotriacetate to the culture medium. Twenty-four hours after iron treatment, a significant increase in aspartate aminotransferase and lactate dehydrogenase in the culture medium was observed. This corresponded to intracellular decrease in the activity of these two enzymes and correlated with a decrease in albumin secretion and an increase in total free malondialdehyde production. The iron toxicity was inhibited by desferrioxamine B. Pyoverdin Pf added to the hepatocyte cultures served as an effective agent to prevent iron toxicity induced in overload. The observed effect of the pyoverdin Pf was as potent as that of desferrioxamine B.

Kinetic evaluation of free malondialdehyde and enzyme leakage as indices of iron damage in rat hepatocyte cultures. Involvement of free radicals.

The present study relates to the effect of ferric iron supplementation on lipid peroxidation of adult rat hepatocyte pure cultures. Lipid peroxidation was evaluated by free malondialdehyde (MDA) using size exclusion chromatography (HPLC) as a specific and sensitive method. The ferric iron used under its complexed form with nitrilotriacetic acid (NTA) exhibited a prooxidant activity corresponding to an increase of free MDA recovery in the cells and in the culture medium. This enhancement of lipid peroxidation in the hepatocyte cultures supplemented with ferric iron was correlated with an intracellular enzyme leakage (lactate dehydrogenase and transaminase), suggesting that lipid peroxidation and enzyme release represented good parameters for cytotoxicity evaluation. The toxic effect of Fe-NTA on hepatocyte cultures was a function of the incubation time (from 0 to 48 hr) and of the concentration of ferric iron loading (i.e. 5, 20 and 100 microM). The mechanism by which Fe-NTA induced cellular damage involved free radical production, as increasing amounts of free radical scavengers corresponded to diminishing rates of both total free MDA and enzyme release. However, this reducing capacity varied from one scavenger to another, where they exhibited preferentially a decrease in lipid peroxidation or in enzyme leakage. This suggested a dissociation between the two parameters of cytotoxicity considered. Lipid peroxidation corresponding to alterations of both inner membranes and the plasma membrane, whereas enzyme release mainly corresponded to the damage of plasma membrane. Subsequently, some scavengers (superoxide dismutase, mannitol, alpha tocopherol, beta carotene) presented an intracellular activity, as they reduced mostly lipid peroxidation. Other ones (catalase, dimethylpyrroline N-oxide, thiourea) seemed essentially efficient in protecting the external plasma membrane, as shown an important decrease in enzyme leakage.

Antioxidant and iron-chelating activities of the flavonoids catechin, quercetin and diosmetin on iron-loaded rat hepatocyte cultures.

The cytoprotective effect of three flavonoids, catechin, quercetin and diosmetin, was investigated on iron-loaded hepatocyte cultures, considering two parameters: the prevention of iron-increased lipid peroxidation and the inhibition of intracellular enzyme release. These two criteria of cytoprotection allowed the calculation of mean inhibitory concentrations (IC50) which revealed that the effectiveness of these flavonoids could be classified as follows: catechin > quercetin > diosmetin. These IC50 values have been related to structural characteristics of the flavonoids tested. Moreover, the investigation of the capacity of these flavonoids to remove iron from iron-loaded hepatocytes revealed a good relationship between this iron-chelating ability and the cytoprotective effect. The cytoprotective activity of catechin, quercetin and diosmetin could thus be ascribed to their widely known antiradical property but also to their iron-chelating effectiveness. These findings increase further the prospects for the development and clinical application of these potent antioxidants.

Antiproliferative effect of deferiprone on the Hep G2 cell line.

Iron is an essential element in cellular metabolism and the growth of all living species, and is involved in DNA replication. The risk of hepatocellular carcinoma development is associated with an increase in iron availability. The aim of the present work was to investigate the effect of an oral iron chelator, deferiprone (CP20), on HepG2 cell-line proliferation in culture. HepG2 cell cultures were maintained in the absence of fetal calf serum (FCS) and in the presence or not (control cultures) of CP20 at the concentrations of 50 or 100 microM; deferoxamine (DFO) was used as an iron chelator reference. Cell proliferation was investigated by the analysis of DNA synthesis using [3H] methyl-thymidine incorporation and of the cell cycle by flow cytometry. Iron chelation efficiency in the culture model was studied by analyzing the effect of CP20 on radioactive iron uptake, intracellular ferritin level, and transferrin receptor expression. CP20, at the concentration of 50 or 100 microM, inhibited DNA synthesis after 48 hr of incubation and induced an accumulation of the cells in the S phase of the cell cycle. Iron chelators inhibited cellular iron uptake, decreased intracellular ferritin level, and increased transferrin receptor protein and mRNA levels. Our results show that CP20 as well as deferoxamine inhibit HepG2 cell proliferation and block cell cycle in the S phase.

New 8-hydroxyquinoline and catecholate iron chelators: influence of their partition coefficient on their biological activity.

Four new hexadendate chelators, three hydroxyquinoline-based, Csox, O-Trensox, Cox750, and one catecholate-based CacCam-which have comparable skeletal structures and pFe, but widely different partition coefficients, (Kpart), 0.01, 0.02, 1 and 3.2 respectively, have been tested for their iron chelating efficacy in vitro by two methods. First, by their ability to remove iron from ferritin in solution or second, to remove iron from iron-loaded hepatocytes in vitro. Our objective was to ascertain the importance of Kpart and pFe, on the biological efficiency of the molecule. Previous studies proposed that an ideal value of Kpart of 1 should give maximum biological activity. Mobilization of iron by Csox and CacCAM from ferritin was similar and furthermore more efficient than desferrioxamine B. In the iron-loaded hepatocyte cultures, the three hydroxyquinoline chelators, although showing diversity in terms of lipophilicity, appeared to be very similar in their capacity to chelate iron. CacCAM, the unique catecholate, was the most efficient of the molecules tested, as well as being the least toxic in the cellular model despite having the lowest value of pFe. In conclusion, the use of the partition coefficient and pFe, as tools for predicting biological activity of iron chelators should be not generalized. Further studies are required in order to understand the influence of the structure on the biological activity of the molecule.

Iron mobilisation and cellular protection by a new synthetic chelator O-Trensox.

We tested a new synthetic, 8-hydroxyquinoline-based, hexadentate iron chelator, O-Trensox and compared it with desferrioxamine B (DFO). Iron mobilisation was evaluated: (i) in vitro by using ferritin and haemosiderin; DFO mobilised iron much more rapidly from ferritin at pH 7.4 than did O-Trensox, whereas at pH 4, ferritin and haemosiderin iron mobilisation was very similar with both chelators; (ii) in vitro by using cultured rat hepatocytes which had been loaded with 55Fe-ferritin; here DFO was slightly more effective after 100 hr than O-Trensox; (iii) in vivo administration i.p. to rats which had been iron-loaded with iron dextran; O-Trensox mobilised 51.5% of hepatic iron over two weeks compared to 48.8% for DFO. We also demonstrated the effect of O-Trensox in decreasing the entry of 55Fe citrate into hepatocyte cultures. The protective effect of O-Trensox against iron toxicity induced in hepatocyte cultures by ferric citrate was shown by decreased release of the enzymes lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotranferase (ALT) from the cultures and, using electron paramagnetic resonance (EPR) measurements, decreased production of lipid radicals. O-Trensox was more effective than DFO in quenching hydroxyl radicals in an acellular system.

Irniine, a pyrrolidine alkaloid, isolated from Arisarum vulgare can induce apoptosis and/or necrosis in rat hepatocyte cultures.

The effects of irniine, a pyrrolidine alkaloid extracted from the tubers of Arisarum vulgare, on rat hepatocyte primary cultures and rat liver epithelial cell line (RLEC) were studied. Cytotoxicity was first evaluated by LDH release, MTT and NR tests and MDA production, while cellular alterations were visualized by electron microscopy and DNA gel-electrophoresis. In hepatocyte and RLEC cultures, a major toxicity appeared at 40 microM of irniine and was demonstrated by an increase in LDH release and decreases in MTT reduction and NR uptake while concentrations lower than 40 microM did not induce significant changes in these parameters. However, we observed an increase in MDA production at 30 microM. Important alterations of the nuclei and mitochondria were also visualized by electron microscopy in cells treated with 50 microM. Using DNA gel-electrophoresis, we demonstrated that irniine at 40 and 50 microM induced DNA damage. All together these results demonstrate that: (1) Irniine induces a significant hepatotoxicity. (2) Irniine toxicity is not mediated by a metabolic derivative since RLEC, which do not contain a monooxygenase system, were also affected by this compound. (3) Irniine induces a significant DNA damage and oxidative stress which leads to cell death by necrosis and/or by apoptosis. Moreover, our data suggest that the alkaloid irniine contained in A. vulgare may be involved in the toxic symptoms observed after medicinal use or consumption of the plant tubers as food both by humans and animals.

Simultaneous measurements of conjugated dienes and free malondialdehyde, used as a micromethod for the evaluation of lipid peroxidation in rat hepatocyte cultures.

Membrane lipid peroxidation in rat hepatocyte cultures was induced by a 5-h incubation with either ethanol (50 mM) or the chelate iron-nitrilotriacetic acid (Fe-NTA) (100 microM). To test the oxidative stress, two indices were measured simultaneously on the same sample: extracellular free malondialdehyde (MDA) measured by HPLC with a size exclusion column, and conjugated dienes (CD) determined by second derivative spectroscopy. With ethanol, both CD and MDA gave nearly the same values of lipid peroxidation, about 135% of the control value. With Fe-NTA, both indices indicated a higher lipid peroxidation, but the MDA and CD values were different. Iron lipid peroxidation evaluated by free MDA and CD was, 290 and 230%, respectively, of the control. This discrepancy could be ascribed to an increased decomposition of hydroperoxides by iron. In addition, the ratio of cis,trans and trans,trans conjugated dienes, which reflects the cellular redox status, remained unchanged after 5 h of lipid peroxidation induced either by ethanol or iron.

Bgugaine, a pyrrolidine alkaloid from Arisarum vulgare, is a strong hepatotoxin in rat and human liver cell cultures.

Toxicity of bgugaine, a pyrrolidine alkaloid extracted from the tubers of Arisarum vulgare, was studied in three different liver cell culture models: (1) the rat hepatocyte primary culture; (2) a liver epithelial cell line; and (3) the human hepatoblastoma cell line HepG2. Cytotoxicity was evaluated by LDH release, MTT reduction and MDA production. DNA fragmentation was analysed by flow cytometry or DNA gel-electrophoresis. In hepatocyte and epithelial cell cultures, drug toxicity appeared at 30 microM and was evaluated by an increase in LDH release, a decrease in MTT reduction and a higher level of MDA production. Bgugaine concentrations lower than 30 microM did not induce changes in these parameters. In HepG2 cells, bgugaine treatment also induced LDH release at concentrations of 40 and 50 microM. DNA fragmentation, analysed in the HepG2 cell line by flow cytometry, was observed in cultures exposed to 50 microM bgugaine. However, using DNA gel-electrophoresis, we demonstrated that lower bgugaine concentrations (10, 20 and 30 microM) also induced DNA damage. Our results show that: (1) bgugaine induces an important hepatotoxicity; (2) bgugaine toxicity is not mediated by a metabolic derivative; and (3) bgugaine induces a significant DNA damage. Therefore, our data suggest that the alkaloid bgugaine contained in Arisarum vulgarae may be involved in the toxicologic symptoms observed after consumption of this plant tubers by humans and animals.

Cytoprotection and iron mobilization in rat hepatocyte cultures by a new synthetic dihydroxamate chelator.

The cytoprotection and iron mobilization effect of a new dihydroxamate chelator 1,1 bis [(11-N-hydroxy)-2,5,11-triaza-1,6,10-trioxo dodecanyl] ethane or KD was studied in primary rat hepatocyte cultures exposed to iron-citrate. Lactate dehydrogenase (LDH) release and malondialdehyde (MDA) production were measured as indexes of cytotoxicity. Cell viability was evaluated using the [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide] (MTT) reduction test. To demonstrate that this chelator was able to decrease iron uptake or increase iron release from the hepatocytes, labelled cells were obtained by maintaining the cultures in the presence of 0.02 microM 55Fe-citrate. The efficacy of KD was compared to desferrioxamine B (DFO) at stoechiometry concentrations. After 24 h of exposure to 50 microM of iron-citrate, a significant release of LDH and MDA was observed. Cell viability was also significantly decreased. When 100 microM of KD were added at the same time as iron, LDH and MDA release was decreased and cell viability was improved. In the presence of the same chelator concentration, a net decrease of iron uptake by the cells was observed as attested by the low intracellular 55Fe level. Moreover, in the 55Fe loaded hepatocytes, the chelator increased the iron extracellular level indicating its iron release effect from the cells. In all tested experimental conditions, the efficacy of 100 microM of the dihydroxamate chelator KD was close to that of 50 microM of the trihydroxamate chelator DFO. In conclusion, KD is effective at a level comparable to DFO in protecting rat hepatocytes against the toxic effect of iron-citrate by decreasing the uptake of the metal and increasing its release from the cells. This synthetic compound appears to have some potential therapeutical interest and the results obtained encourage the synthesis of new hydroxamate ligands.

Inhibition of iron toxicity in rat hepatocyte culture by natural phenolic compounds.

Iron supplementation of adult rat hepatocyte culture induced a cytotoxic effect as shown by an increase of lipid peroxidation. The antioxidant activity of some natural phenolic compounds from olive oil (caffeic acid, oleuropein, tyrosol and hydroxytyrosol) has been investigated on this iron-loaded hepatocyte culture model. These compounds greatly reduced malondialdehyde production which was used as a marker for iron-induced lipid peroxidation. This reduction was concentration-dependent of phenolic compound (in the range of 20-100 mum). Moreover, it was not significantly different from one tested compound to another. To clarify the antioxidant mechanism of these compounds, their free radical scavenging activity has been tested in a cell-free experimental model using spin trapping-electron paramagnetic resonance spectroscopy. The four tested compounds were able to scavenge hydroxyl and lipid radicals. They exhibited various efficiency towards hydroxyl radical whereas they presented the same order of reactivity towards lipid radicals. Moreover, only caffeic acid and oleuropein could scavenge Superoxide anion. Therefore, the reactivity of the phenolic compounds towards these reactive oxygen species provided an insight into their antioxidant activity in iron-loaded hepatocyte culture. These compounds could probably interfere with the chain-propagating steps of the lipid peroxidation induced by iron in hepatocytes, which resulted in an inhibition of toxicity.