Isabelle Morel

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.

ROLE OF FLAVONOIDS AND IRON CHELATION IN ANTIOXIDANT ACTION

Publisher Summary This chapter discusses the role of flavonoids and iron chelation in antioxidant action. The potential of flavonoids to inhibit lipid peroxidation in biological models is supposed to reside mainly in their free radical scavenging capacity rather than in their iron chelating activity. This property is considered as a minor mechanism in the antioxidant action, because it has not been clearly established in biological systems. The assessment of a relationship between the antioxidant effect and the iron chelating capacity of flavonoids is subsequently of interest. For this purpose, rat is used hepatocyte cultures as a biological model where lipid peroxidation is induced by iron [Fe(III)] in its complexed form with nitrilotriacetic acid (NTA). The Fe-NTA complex is known to induce a rapid accumulation of iron inside the cells. Nitrilotriacetic acid (NTA) is used to maintain ferric iron in a soluble state; it is a low-affinity iron chelator.

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.

Repair of iron-induced DNA oxidation by the flavonoid myricetin in primary rat hepatocyte cultures

Oxidative DNA damage and its repair in primary rat hepatocyte cultures was investigated following 4 h of incubation with the toxic iron chelate, ferric nitrilotriacetate (Fe-NTA), in the presence or absence of the potent protective flavonoid myricetin (25-50-100 microM). Seven DNA base oxidation products were quantified in DNA extracts by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring mode. Concomitantly, DNA repair capacity of hepatocytes was estimated by the release of oxidized-base products into culture media, using the same GC-MS method. A genotoxic effect of Fe-NTA (100 microM) in hepatocytes was evidenced by a severe increase in DNA oxidation over basal levels, with accumulation in cellular DNA of five oxidation products derived from both purines and pyrimidines. This prooxidant effect of iron was also noted by an induction of lipid peroxidation, estimated by free malondialdehyde production. Addition of increasing concentrations of myricetin (25-50-100 microM) simultaneously with iron prevented both lipid peroxidation and accumulation of oxidation products in DNA. Moreover, as an activation of DNA repair pathways, myricetin stimulated the release of DNA oxidation bases into culture media, especially of purine-derived oxidation products. This removal of highly mutagenic oxidation products from DNA of hepatocytes might correspond to an activation of DNA excision-repair enzymes by myricetin. This was verified by RNA blot analysis of DNA polymerase beta gene expression which was induced by myricetin in a dose-dependent manner. This represented a novel and original mechanism of cytoprotection by myricetin against iron-induced genotoxicity via stimulation of DNA repair processes. Since iron-induced DNA damage and inefficient repair in hepatocytes could be related to genotoxicity and most probably to hepatocarcinogenesis, modulation of these processes in vitro by myricetin might be relevant in further prevention of liver cancer derived from iron overload pathologies.

Mechanism of tert-butylhydroperoxide induced apoptosis in rat hepatocytes: involvement of mitochondria and endoplasmic reticulum

The purpose of the present work was to study the mechanisms involved in apoptosis induced by oxidative stress in rat hepatocytes. We focused on the apoptotic signaling molecules cytochrome c, Bcl-2 and Bax. Rat hepatocytes were exposed for 1 h to increasing concentrations of tert-butylhydroperoxide (t-BHP). Using lactate dehydrogenase (LDH) leakage as a biomarker for necrosis, and DNA fragmentation as a biomarker for apoptosis, we observed that a concentration of t-BHP of 0.4-0.5 mM provides a transition point below which apoptosis is favored and beyond which necrosis is favored. Malondialdehyde and 8-oxo-guanine formation indicates that t-BHP induces oxidative stress and damage. However, at 0.4 mM t-BHP, these oxidative molecular changes as well as LDH leakage no longer progress after the first hour of t-BHP exposure, suggesting the activation of some defense mechanisms. Western blot analysis of cytochrome c shows that its level increases in the cytosol while that of Bax decreases in this fraction as a result of t-BHP treatment. Moreover, there is a loss of Bcl-2 from mitochondria while, in contrast, Bax accumulates in this organelle following t-BHP treatment. However, cytochrome c appears to be relocalized to the endoplasmic reticulum as its presence in microsomes is greatly enhanced. We suggest that t-BHP triggers apoptosis through a step that involves cytochrome c release from mitochondria. This event is stimulated by Bcl-2 disappearance from mitochondria and Bax recruitment. Neutralization of excess cytosolic cytochrome c is achieved by its relocalization to the endoplasmic reticulum, hence triggering the down-regulation of apoptotic signals.

Inter-laboratory validation of procedures for measuring 8-oxo-7,8-dihydroguanine/8-oxo-7,8-dihydro-2 '-deoxyguanosine in DNA

The aim of ESCODD, a European Commission funded Concerted Action, is to improve the precision and accuracy of methods for measuring 8-oxo-7,8-dihydroguanine (8-oxoGua) or the nucleoside (8-oxodG). On two occasions, participating laboratories received samples of different concentrations of 8-oxodG for analysis. About half the results returned (for 8-oxodG) were within 20% of the median values. Coefficients of variation (for three identical samples) were commonly around 10%. A sample of calf thymus DNA was sent, dry, to all laboratories. Analysis of 8-oxoGua/8-oxodG in this sample was a test of hydrolysis methods. Almost half the reported results were within 20% of the median value, and half obtained a CV of less than 10%. In order to test sensitivity, as well as precision, DNA was treated with photosensitiser and light to introduce increasing amounts of 8-oxoGua and samples were sent to members. Median values calculated from all returned results were 45.6 (untreated), 53.9, 60.4 and 65.6 8-oxoGua/10 6 Gua; only seven laboratories detected the increase over the whole range, while all but one detected a dose response over two concentration intervals. Results in this trial reflect a continuing improvement in precision and accuracy. The next challenge will be the analysis of 8-oxodG in DNA isolated from cells or tissue, where the concentration is much lower than in calf thymus DNA.

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.

Differences in Early Acetaminophen Hepatotoxicity between Obese ob/ob and db/db Mice

Clinical investigations suggest that hepatotoxicity after acetaminophen (APAP) overdose could be more severe in the context of obesity and nonalcoholic fatty liver disease. The pre-existence of fat accumulation and CYP2E1 induction could be major mechanisms accounting for such hepatic susceptibility. To explore this issue, experiments were performed in obese diabetic ob/ob and db/db mice. Preliminary investigations performed in male and female wild-type, ob/ob, and db/db mice showed a selective increase in hepatic CYP2E1 activity in female db/db mice. However, liver triglycerides in these animals were significantly lower compared with ob/ob mice. Next, APAP (500 mg/kg) was administered in female wild-type, ob/ob, and db/db mice, and investigations were carried out 0.5, 2, 4, and 8 h after APAP intoxication. Liver injury 8 h after APAP intoxication was higher in db/db mice, as assessed by plasma transaminases, liver histology, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. In db/db mice, however, the extent of hepatic glutathione depletion, levels of APAP-protein adducts, c-Jun N-terminal kinase activation, changes in gene expression, and mitochondrial DNA levels were not greater compared with the other genotypes. Furthermore, in the db/db genotype plasma lactate and β-hydroxybutyrate were not specifically altered, whereas the plasma levels of APAP-glucuronide were intermediary between wild-type and ob/ob mice. Thus, early APAP-induced hepatotoxicity was greater in db/db than ob/ob mice, despite less severe fatty liver and similar basal levels of transaminases. Hepatic CYP2E1 induction could have an important pathogenic role when APAP-induced liver injury occurs in the context of obesity and related metabolic disorders.

Dopexamine and norepinephrine versus epinephrine on gastric perfusion in patients with septic shock: a randomized study [NCT00134212]

IntroductionMicrocirculatory blood flow, and notably gut perfusion, is important in the development of multiple organ failure in septic shock. We compared the effects of dopexamine and norepinephrine (noradrenaline) with those of epinephrine (adrenaline) on gastric mucosal blood flow (GMBF) in patients with septic shock. The effects of these drugs on oxidative stress were also assessed.MethodsThis was a prospective randomized study performed in a surgical intensive care unit among adults fulfilling usual criteria for septic shock. Systemic and pulmonary hemodynamics, GMBF (laser-Doppler) and malondialdehyde were assessed just before catecholamine infusion (T0), as soon as mean arterial pressure (MAP) reached 70 to 80 mmHg (T1), and 2 hours (T2) and 6 hours (T3) after T1. Drugs were titrated from 0.2 μg kg-1 min-1 with 0.2 μg kg-1 min-1 increments every 3 minutes for epinephrine and norepinephrine, and from 0.5 μg kg-1 min-1 with 0.5 μg kg-1 min-1 increments every 3 minutes for dopexamine.ResultsTwenty-two patients were included (10 receiving epinephrine, 12 receiving dopexamine–norepinephrine). There was no significant difference between groups on MAP at T0, T1, T2, and T3. Heart rate and cardiac output increased significantly more with epinephrine than with dopexamine–norepinephrine, whereas. GMBF increased significantly more with dopexamine–norepinephrine than with epinephrine between T1 and T3 (median values 106, 137, 133, and 165 versus 76, 91, 90, and 125 units of relative flux at T0, T1, T2 and T3, respectively). Malondialdehyde similarly increased in both groups between T1 and T3.ConclusionIn septic shock, at doses that induced the same effect on MAP, dopexamine–norepinephrine enhanced GMBF more than epinephrine did. No difference was observed on oxidative stress.

Involvement of Phenoxyl Radical Intermediates in Lipid Antioxidant Action of Myricetin in Iron-Treated Rat Hepatocyte Culture

Supplementation of rat hepatocyte cultures with the flavonoid myricetin (300 microM) led to the formation of phenoxyl radical intermediates, as detected in intact cells by electron paramagnetic resonance (EPR) spectroscopy. These radicals corresponded to one-electron oxidation products of myricetin. The level of phenoxyl radicals was significantly reduced when myricetin-treated hepatocyte cultures were also supplemented with iron (Fe-NTA 100 microM). This suggested that iron could accelerate the oxidation flux of myricetin. Moreover, myricetin was found to be able to inhibit lipid peroxidation induced by iron in hepatocyte culture. Free malondialdehyde (MDA) levels and the amount of radicals derived from oxidized lipids were greatly reduced when myricetin was added to iron-treated cultures. This showed that myricetin was a good inhibitor of lipid peroxidation in this model and that the intermediate generation of phenoxyl radicals might contribute to the antioxidant mechanism of myricetin.