Exercise-induced oxidative stress in overweight adolescent girls: roles of basal insulin resistance and inflammation and oxygen overconsumption.

HYPOTHESIS: Basal insulin resistance (IR) and inflammation exacerbate post-exercise oxidative stress (OS) in overweight adolescent girls. DESIGN: Cross-sectional study, effect of incremental ergocycle exercise until exhaustion on OS markers. PARTICIPANTS: Normal-weight (control) (n=17, body mass index (BMI): 20-24.2 kg/m(2)) and overweight adolescent girls (n=29, BMI: 24.1-36.6 kg/m(2)). MEASUREMENTS: Dietary measurement, physical activity assessment (validated questionnaires), fat distribution parameters (by dual-energy X-ray absorptiometry and anthropometry) and maximal oxygen consumption (VO2peak). Blood assays include the following: (1) at fasting state: blood cell count, lipid profile, and IR parameters (leptin/adiponectin ratio (L/A), homeostasis model assessment of IR, insulin/glucose ratio; (2) before exercise: inflammation and OS markers (interleukin-6 (IL-6), C-reactive protein (CRP), myeloperoxidase (MPO), reduced glutathione/oxidized glutathione ratio (GSH/GSSG), 15 F(2)alpha-isoprostanes (F(2)-Isop), lipid hydroperoxides (ROOH), oxidized low-density lipoprotein (ox-LDL)) and antioxidant status (superoxide dismutase (SOD), glutathione peroxidase (GPX), vitamin C, alpha-tocopherol and beta-carotene); and (3) after exercise: inflammation and OS markers. RESULTS: At rest, overweight girls had a deteriorated lipid profile and significantly higher values of IR parameters and inflammation markers, compared with the control girls. These alterations were associated with a moderate rest OS state (lower GSH/GSSG ratio, alpha-tocopherol/total cholesterol (TC) ratio and GPX activity). In absolute values, overweight girls exhibited higher peak power output and oxygen consumption (VO2peak), compared with the control girls. Exercise exacerbated OS only in the overweight group (significant increase in F(2)-Isop, ROOH and MPO). As hypothesized, basal IR and inflammation state were correlated with the post-exercise OS. However, the adjustment of F(2)-Isop, ROOH and MPO variation per exercise VO(2) variation canceled the intergroup differences. CONCLUSION: In overweight adolescent girls, the main factors of OS, after incremental exhaustive exercise, are not the basal IR and inflammation states, but oxygen overconsumption.

Selective inhibition of NF-kappaB activation by the flavonoid hepatoprotector silymarin in HepG2. Evidence for different activating pathways.

The bioflavonoid silymarin is found to potently suppress both nuclear factor kappa-B (NF-kappaB)-DNA binding activity and its dependent gene expression induced by okadaic acid in the hepatoma cell line HepG2. Surprisingly, tumor necrosis factor-alpha-induced NF-kappaB activation was not affected by silymarin, thus demonstrating a pathway-dependent inhibition by silymarin. Many genes encoding the proteins of the hepatic acute phase response are under the control of the transcription factor NF-kappaB, a key regulator in the inflammatory and immune reactions. Thus, the inhibitory effect of silymarin on NF-kappaB activation could be involved in its hepatoprotective property.

Dexamethasone differently modulates TNF-alpha- and IL-1beta-induced transcription of the hepatic Mn-superoxide dismutase gene.

The effects of cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and the synthetic glucocorticoid dexamethasone on the gene expression of antioxidant enzymes have been investigated in rat hepatocytes in primary culture. First, we observed that the hepatocyte culture process induced a strong but transient induction of manganese superoxide dismutase (Mn-SOD) gene expression, whereas copper-zinc superoxide dismutase, glutathione peroxidase and catalase genes were down-regulated. IL-1beta and TNF-alpha both stimulated specifically Mn-SOD gene expression in a time-dependent manner. TNF-alpha rapidly induced Mn-SOD gene expression while IL-1beta was a strong but slow inducer of this gene. Both cytokines acted at the transcriptional level as shown by nuclear run on assays. Dexamethasone prevented the TNF-alpha- but not the IL-1beta induced up-regulation of Mn-SOD gene transcription by a mechanism likely to involve the glucocorticoid receptor. Moreover this glucocorticoid did not suppress the TNF-alpha-induced increase of NF-kappaB binding activity. These results suggest that IL-1beta and TNF-alpha regulate Mn-SOD gene transcription by different pathways.

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.

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.

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.

Antioxidants modulate acute solar ultraviolet radiation-induced NF-kappa-B activation in a human keratinocyte cell line.

Exposure of the human skin to ultraviolet radiation (UVR) leads to depletion of cutaneous antioxidants, regulation of gene expression and ultimately to the development of skin diseases. Although exogenous supplementation of antioxidants prevents UVR-induced photooxidative damage, their effects on components of cell signalling pathways leading to gene expression has not been clearly established. In the present study, the effects of the antioxidants alpha-lipoic acid, N-acetyl-L-cysteine (NAC) and the flavonoid extract silymarin were investigated for their ability to modulate the activation of the transcription factors nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1) in HaCaT keratinocytes after exposure to a solar UV simulator. The activation of NF-kappaB and AP-1 showed a similar temporal pattern: activation was detected 2 h after UV exposure and maintained for up to 8 h. To determine the capacity of activated NF-kappaB to stimulate transcription, NF-kappaB-dependent gene expression was measured using a reporter gene assay. The effects of the antioxidants on NF-kappaB and AP-1 activation were evaluated 3 h after exposure. While a high concentration of NAC could achieve a complete inhibition, low concentrations of alpha-lipoic acid and silymarin were shown to significantly inhibit NF-kappaB activation. In contrast, AP-1 activation was only partially inhibited by NAC, and not at all by alpha-lipoic acid or silymarin. These results indicate that antioxidants such as alpha-lipoic acid and silymarin can efficiently modulate the cellular response to UVR through their selective action on NF-kappaB activation.

Comparison of oxidative damage of DNA and lipids in normal and tumor rat hepatocyte cultures treated with ferric nitrilotriacetate.

Oxidative damage of DNA and lipids in normal primary rat hepatocyte cultures and in hepatoma Fao cell-line was induced by ferric nitrilotriacetate (Fe-NTA). DNA oxidation was evidenced by measuring the mutagenic oxidized nucleoside 8-hydroxy-2'-deoxyguanosine (8-oxodG). An increase in 8-oxodG production was induced by Fe-NTA in the two different cell cultures. Moreover, this increase was more important in hepatocytes than in Fao cells. In addition, the extent of lipid peroxidation was higher in normal hepatocytes than in Fao cells. These observations demonstrated a higher resistance of tumor cells than normal hepatocytes to oxidative stress. Since DNA lesions induced by oxidative stress are now recognized as being involved in the mutagenesis process and since normal hepatocytes appeared particularly sensitive to iron-induced oxidative damage, a high level of iron should be considered as a potent toxic factor involved in normal cell degeneration. This findings might partly explain the propensity of hepatic iron-overload diseases for cancerous evolution.

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.

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.

Free radical scavenging and antioxidant effects of lactate ion: an in vitro study.

Divergent literature data are found concerning the effect of lactate on free radical production during exercise. To clarify this point, we tested the pro- or antioxidant effect of lactate ion in vitro at different concentrations using three methods: 1) electron paramagnetic resonance (EPR) was used to study the scavenging ability of lactate toward the superoxide aion (O(2)(-).) and hydroxyl radical (.OH); 2) linoleic acid micelles were employed to investigate the lipid radical scavenging capacity of lactate; and 3) primary rat hepatocyte culture was used to study the inhibition of membrane lipid peroxidation by lactate. EPR experiments exhibited scavenging activities of lactate toward both O(2)(-). and.OH; lactate was also able to inhibit lipid peroxidation of hepatocyte culture. Both effects of lactate were concentration dependent. However, no inhibition of lipid peroxidation by lactate was observed in the micelle model. These results suggested that lactate ion may prevent lipid peroxidation by scavenging free radicals such as O(2)(-). and.OH but not lipid radicals. Thus lactate ion might be considered as a potential antioxidant agent.

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.

The relationship between fatty acid peroxidation and alpha-tocopherol consumption in isolated normal and transformed hepatocytes.

The response of normal and transformed rat hepatocytes to oxidative stress was investigated. Isolated normal rat hepatocytes and differentiated hepatoma cells (the Fao cell line was derived from the Reuber H 35 rat hepatoma) in suspension were incubated with the ADP/Fe3+ chelate for 30 min at 37 degrees C. Membrane lipid oxidation was assessed by measuring (i) free malondialdehyde (MDA) production by a high-performance liquid chromatography (HPLC) procedure, (ii) membrane fatty acid disappearance as judged by capillary gas chromatography, and (iii) alpha-tocopherol oxidation as determined by HPLC and electrochemical detection. The addition of iron led to increased MDA production in normal as well as in transformed cells, and to simultaneous consumption of polyunsaturated fatty acids (PUFA) and alpha-tocopherol. In addition, in Fao cells more alpha-tocopherol was consumed during lipid peroxidation while less PUFA was oxidized. Lipid peroxidation was lower in tumoral hepatocytes than in normal cells. This could be due to a difference in membrane lipid composition because of a lower PUFA content and a higher alpha-tocopherol level in Fao cells. During oxidation, Fao cells produced 1.5 to 2 times less MDA than normal cells, while in the tumoral cells the amount of oxidized PUFA having 3 or more double bonds was 7 to 8 times lower. Therefore, measuring MDA alone as an index of lipid peroxidation did not allow for proper comparison of the membrane lipid oxidizability of transformed cells vs. the membrane lipid oxidizability of normal cells.

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 (< or = 1 hr) was observed in ethanol-supplemented cultures; it was inhibited by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, whereas alpha-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.

Physical inactivity, insulin resistance, and the oxidative-inflammatory loop.

Epidemiological data indicate that physical inactivity, a main factor of global energetic imbalance, is involved in the worldwide epidemic of obesity and metabolic disorders such as insulin resistance. Although the complex pathogenesis of insulin resistance is not fully understood, literature data accumulated during the past decades clearly indicate that the activation of the oxidative-inflammatory loop plays a major role. By activating the oxidative-inflammatory loop in insulin-sensitive tissues, fat gain and adipose tissue dysfunction likely contribute to induce insulin resistance during chronic and prolonged physical inactivity. However, in the past years, evidence has emerged showing that early insulin resistance also occurs after very short-term exposure to physical inactivity (1-7 days) without any fat gain or energetic imbalance. The possible role of liver disturbances or endothelial dysfunction is suggested, but further studies are necessary to really conclude. Inactive skeletal muscle probably constitutes the primary triggering tissue for the development of early insulin resistance. In the present review, we discuss on the current knowledge about the effect of physical inactivity on whole-body and peripheral insulin sensitivity, and how local inflammation and oxidative stress arising with physical inactivity could potentially induce insulin resistance. We assume that early muscle insulin resistance allows the excess nutrients to shift in the storage tissues to withstand starvation through energy storage. We also consider when chronic and prolonged, physical inactivity over an extended period of time is an underestimated contributor to pathological insulin resistance and hence indirectly to numerous chronic diseases.