Nicole Pasdeloup

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.

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 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.

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

Abstract.  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 G2‐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 G2‐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 N1‐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.

INCREASE IN CELLULAR POOL OF LOW-MOLECULAR-WEIGHT IRON DURING ETHANOL METABOLISM IN RAT HEPATOCYTE CULTURES : RELATIONSHIP WITH LIPID PEROXIDATION

Ethanol-induced lipid peroxidation was studied in primary rat hepatocyte cultures supplemented with ethanol at the concentration of 50 mM. Lipid peroxidation was assessed by two indices: (1) conjugated dienes by second-derivative UV spectroscopy in lipid extract of hepatocytes (intracellular content), and (2) free malondialdehyde (MDA) by HPLC-UV detection and quantitation for the incubation medium (extracellular content). In cultures supplemented with ethanol, free MDA increased significantly in culture media, whereas no elevation of conjugated diene level was observed in the corresponding hepatocytes. The cellular pool of low-mol-wt (LMW) iron was also evaluated in the hepatocytes using an electron spin resonance procedure. An early increase of intracellular LMW iron (≤1 hr) was observed in ethanol-supplemented cultures; it was inhibited by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, whereas α-tocopherol, which prevented lipid peroxidation, did not inhibit the increase of LMW iron. Therefore, the LMW iron elevation was the result of ethanol metabolism and was not secondarily induced by lipid hydroperoxides. Thus, ethanol caused lipid peroxidation in rat hepatocytes as shown by the increase of free MDA, although no conjugated diene elevation was detected. During ethanol metabolism, an increase in cellular LMW iron was observed that could enhance conjugated diene degradation.

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.

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.

Inhibition of iron toxicity in rat and human hepatocyte cultures by the hydroxypyridin-4-ones CP20 and CP94

The protective effect of the hydroxypyridin-4-ones (CP20 and CP94) was studied on iron-loaded rat and human hepatocytes; desferrioxamine B was used as a chelator reference. Iron load was achieved by addition of 5 up to 50 microM iron citrate to the culture medium. One day after iron treatment, an increase in lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and malondialdehyde extracellular concentrations was measured in rat and human hepatocyte cultures. This enzyme release and the increase in free extracellular malondialdehyde were observed with 5 microM iron and high levels were obtained with 50 microM. The bidentate chelators CP20 and CP94 (150 microM) appeared to be as effective as the hexadentate chelator desferrioxamine (50 microM) in the protection of rat and human hepatocytes against the toxic effect of iron load achieved by culturing the cells for 1 day in the presence of 50 microM iron citrate. In rat and human hepatocytes cultured for 1 day in the presence of 1 microM 55Fe-50 microM iron citrate plus CP20, CP94 or desferrioxamine B, a decrease of iron uptake by the cells was observed. When the hepatocytes were cultured for 1 day in the presence of 1 microM 55Fe-50 microM iron citrate and then for a further day in the presence of CP20, CP94 or desferrioxamine B but not iron, the chelators decreased the intracellular iron level, indicating their iron releasing effect from the loaded cells. The observed effects of the hydroxypyridin-4-ones CP20 and CP94 were as potent as the effect of desferrioxamine B. This study presents new data favoring the potential clinical interest of this new class of chelating agents in the treatment of human iron overload.

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.

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.