Biomedical subjects

S Puntarulo

Publications and source records attributed to S Puntarulo.

At least 19 recordsLinked to original sources

PubMed2003

Production of reactive oxygen species by isolated mitochondria of the Antarctic bivalve Laternula elliptica (King and Broderip) under heat stress.

Formation of reactive oxygen species (ROS) in mitochondrial isolates from gill tissues of the Antarctic polar bivalve Laternula elliptica was measured fluorimetrically under in vitro conditions. When compared to the rates measured at habitat temperature (1 degrees C), significantly elevated ROS formation was found under temperature stress of 7 degrees C and higher. ROS formation correlated significantly with oxygen consumption in individual mitochondrial preparations over the entire range of experimental temperatures (1-12 degrees C). ROS generation per mg of mitochondrial protein was significantly higher in state 3 at maximal respiration and coupling to energy conservation, than in state 4+, where ATPase-activity is inhibited by oligomycin and only proton leakage is driving the residual oxygen consumption. The percent conversion of oxygen to the membrane permeant hydrogen peroxide amounted to 3.7% (state 3) and 6.5% (state 4+) at habitat temperature (1 degrees C), and to 7% (state 3) and 7.6% (state 4+) under experimental warming to 7 degrees C. This is high compared to 1-3% oxygen to ROS conversion in mammalian mitochondrial isolates and speaks for a comparatively low control of toxic oxygen formation in mitochondria of the polar bivalve. However, low metabolic rates at cold Antarctic temperatures keep absolute rates of mitochondrial ROS production low and control oxidative stress at habitat temperatures. Mitochondrial coupling started to fall beyond 3 degrees C, closely to pejus temperature (4 degrees C) of the bivalve. Accordingly, the proportion of state 4 respiration increased from below 30% at 1 degrees C to over 50% of total oxygen consumption at 7 degrees C, entailing reduced ADP/O ratios under experimental warming. Progressive mitochondrial uncoupling and formation of hazardous ROS contribute to bias mitochondrial functioning under temperature stress in vitro. Deduced from a pejus temperature, heat stress commences already at 5 degrees C, and is linked to progressive loss of phosphorylation efficiency, increased mitochondrial oxygen demand and elevated oxidative stress above pejus temperatures.

Adenosine Triphosphatases↗

PubMed2002

Temperature-dependence of mitochondrial function and production of reactive oxygen species in the intertidal mud clam Mya arenaria.

Mitochondrial respiration, energetic coupling to phosphorylation and the production of reactive oxygen species (ROS) were studied in mitochondria isolated from the eurythermal bivalve Mya arenaria (Myoidea) from a low-shore intertidal population of the German Wadden Sea. Measurements were conducted both within the range of the habitat temperatures (5-15 degrees C) and when subjected to heat exposure at 20 degrees C and 25 degrees C. Experimental warming resulted in an increase in the rate of state 3 and state 4 respiration in isolated mitochondria. The highest respiratory coupling ratios (RCR) were found at 15 degrees C; at higher temperatures mitochondrial coupling decreased, and release of ROS doubled between 15 and 25 degrees C. ROS production was 2-3% of total oxygen consumption in state 3 (0.3-0.5 nmol ROS mg(-1) protein min(-1)) at the habitat temperature, reaching a maximum of 4.3 % of state 3 respiration and 7 % of oligomycin-induced state 4+ respiration under heat stress. Thus, state 4 respiration, previously interpreted exclusively as a measure of proton leakage, included a significant contribution from ROS formation in this animal, especially under conditions of heat stress. Oxygen radical formation was directly dependent on temperature-controlled respiration rates in states 3 and 4 and inversely related to mitochondrial coupling (RCR+) in state 4. Mitochondrial ROS formation is therefore involved in cellular heat stress in this eurythermal marine ectotherm.

Animals↗

PubMed2001

Nitric oxide and iron overload. Limitations of ESR detection by DETC.

The ability of the ESR technique based on diethyldithiocarbamate (DETC) administration was studied as a suitable method to assess NO generation in vivo. The technique was successfully employed to measure NO generation after LPS treatment. DETC2-Fe-NO adducts were detected in liver homogenates of iron overloaded animals. When iron was administered to the animals simultaneously with LPS, NO-dependent signal increased 122%, but the content of NO2- and NO3- in sera was significantly lower (44%) as compared to LPS-treated rats. Iron dextran administration was responsible for a three-fold increase in the DETC2-Fe-NO content in non-LPS treated rats, while NOS activity and sera NO2- and NO3- levels remained unaffected. The adduct generation rate by a chemical NO-source was recorded in the presence of either control or iron overloaded homogenates supplemented with DETC in vivo. The exposure of liver homogenates to NO was performed either by the addition of 1 mM SNAP as NO donor or infusing an aqueous NO solution. In the presence of iron overloaded samples the adduct generation rate was 3.8-4.4-fold higher than in the presence of control samples. This effect restricts the applicability of the method to experimental conditions where iron levels remain constant, therefore it is not suitable for NO generation studies in experimental models where animals were subjected to iron overload.

Animals↗

PubMed2001

UV-B effects on Antarctic Chlorella sp cells.

Growth of Antarctic Chlorella sp cells was measured in cultures irradiated with 30 kJ m(-2) UV-B (280-320 nm). The specific growth rate immediately after the lag phase was 0.36+/-0.06 and 0.26+/-0.03 day(-1) for unirradiated cultures and cultures irradiated with UV-B, respectively, UV-B irradiation significantly decreased ascorbate content by 54.5%, and increased the ascorbyl radical content/ascorbate content ratio by 2.25-fold in algae cultures in log phase. UV-B exposure significantly decreased by 95, 62 and 71% the content of alpha-tocopherol, beta-carotene and total thiols, respectively, in cells in log phase of development. The cellular content of alpha-tocopherol, beta-carotene and total thiols was reduced by 78, 43 and 44%, respectively in stationary phase, as compared to the antioxidant content in the cells during log phase of development. UV-B exposure reduced the content of alpha-tocopherol and total thiols in stationary phase of development by 64 and 91%, respectively, as compared to unirradiated cells. The content of beta-carotene in stationary phase was not affected by UV-B exposure. The results presented here suggest that increased UV-B radiation was responsible for the development of oxidative stress conditions, assessed as the ascorbyl radical content/ascorbate content ratio, in Antarctic Chlorella sp cells. Moreover, a significant decrease in the content of both lipid and water soluble antioxidants might contribute to establish oxidative stress in the cells.

Antarctic Regions↗

PubMed2001

Iron-induced changes in nitric oxide and superoxide radical generation in rat liver after lindane or thyroid hormone treatment.

The involvement of cytosolic nitric oxide (NO) and mitochondrial superoxide radical (O2(.-)) production was evaluated as a mechanism triggering liver oxidative stress in lindane (40 mg/kg) or L-3,3',5-triiodothyronine (T3, 0.1 mg/kg for 2 consecutive days) treated animals (male Sprague-Dawley rats) subjected to iron overload (200 mg/kg). Lindane and iron led to 504 and 210% increases in the content of hepatic protein carbonyls as an index of oxidative stress, with a 706% enhancement being produced by their combined administration. T3 did not alter this parameter, whereas iron overload increased the content of protein carbonyls by 116% in hyperthyroid rats. Lindane increased NO generation by 106% without changes in generation of O2(.-), whereas iron enhanced both parameters by 109 and 80% over control values, respectively, with a net 33 and 46% decrease, respectively, being elicited by the combined treatment related to iron overload alone. Hyperthyroidism increased liver NO (69%) and O2(.-) (110%) generation compared to controls, effects that were either synergistically augmented or suppressed by iron overload, respectively. The in vitro addition of iron (1 micromol/mg protein) to liver cytosolic fractions from euthyroid (97%) and hyperthyroid (173%) rats also enhanced NO generation. The effects of iron overload on mitochondrial O2(.-) production by hyperthyroid rats were reproduced by the in vitro addition of 1 micromol iron/mg protein and abolished by the in vivo pretreatment with the iron chelator desferrioxamine (500 mg/kg). It is concluded that liver oxidative stress induced by iron overload is independent of NO and O2(.-) production in lindane-treated rats, whereas in hyperthyroid animals NO generation is a major factor contributing to this redox imbalance.

Animals↗

PubMed2001

Effects of iron overload and lindane intoxication in relation to oxidative stress, Kupffer cell function, and liver injury in the rat.

Parameters related to liver oxidative stress, Kupffer cell function, and hepatocellular injury were assessed in control rats and in animals subjected to lindane (40 mg/kg; 24 h) and/or iron (200 mg/kg; 4 h) administration. Independently of lindane treatment, iron overload enhanced the levels of iron in serum and liver. Biliary efflux of glutathione disulfide increased by 140, 160, or 335% by lindane, iron, or their combined administration, respectively, and the hepatic content of protein carbonyls was elevated by 5.84-, 2.95-, and 10-fold. Colloidal carbon uptake by perfused livers was not modified by lindane and/or iron, whereas gadolinium chloride (GdCl(3)) pretreatment diminished uptake by 60-72%. Carbon-induced liver O(2) uptake was not altered by lindane, whereas iron produced a 61% increase and the combined treatment led to a 72% decrease over control values. Pretreatment with GdCl(3) abolished these effects in all groups. Lindane-treated rats showed acidophilic hepatocytes in periportal areas and some hepatic cells with nuclear pyknosis, whereas iron overload led to moderate hyperplasia and hypertrophy of Kupffer cells and moderate inflammatory cell infiltration. Combined lindane-iron treatment led to hepatocytes with pyknotic nuclei, significant acidophilia, and extensive lymphatic and neutrophil infiltration in the portal space. Hepatic myeloperoxidase activity increased by 1.1-, 2.1-, or 6.7-fold by lindane, iron, or their combined administration, respectively. Liver sinusoidal lactate dehydrogenase efflux increased by 2.2-fold (basal conditions) and 9.7-fold (carbon infusion) in the lindane-iron treated rats, effects that were diminished by 35 and 78% by GdCl(3) pretreatment, respectively. These data support the contention that lindane sensitizes the liver to the damaging effects of iron overload by providing an added enhancement to the oxidative stress status in the tissue, and this may contribute to the alteration of the respiratory activity of Kupffer cells and the development of an inflammatory response.

Animals↗

PubMed2001

Antioxidant capacity of a 3-deoxyanthocyanidin from soybean.

Soybean cotyledons directly exposed to UV-C (190-280 nm) contained a colored pigment in those areas of the epidermis directly exposed to UV-C. Ethanolic extracts from UV-C irradiated cotyledons showed a significant peak at 532 nm at pH=10, but not seen at pH=6, successive changes in pH were accompanied by reversible changes in the spectra. The identity of the pigment isolated from soybean cotyledons was established as apigeninidin by comparing the features of standard of a apigeninidin (from sorghum) previously characterized by FAB-MS, UV, HPLC, 1H NMR, and IR spectroscopy. To characterize antioxidant activity of this compound, its ability to scavenge radical species in vitro was tested. In the concentration range tested (up to 200 microg ml (-1)), apigeninidin did not show any scavenger activity towards hydroxyl radical, quinones or NO. However, ascorbyl radical and lipid radicals were effectively quenched in a dose-dependent manner. Overall, UV-C radiation triggers molecular signals that lead in soybean cotyledons to the synthesis and accumulation of an antioxidant pigment, apigeninidin, that shows scavenger activity against ascorbyl and lipid radicals in in vitro studies.

Anthocyanins↗

PubMed1999

Interaction of 1-hydroxyethyl radical with antioxidant enzymes.

There is considerable interest in the role of the 1-hydroxyethyl radical (HER) in the toxic effects of ethanol. The goal of this study was to evaluate the effects of HER on classical antioxidant enzymes. The interaction of acetaldehyde with hydroxylamine-o-sulfonic acid has been shown to produce 1, 1'-dihydroxyazoethane (DHAE); this compound appears to be highly unstable, and its decomposition leads to the generation of HER. Addition of DHAE into a solution of PBN led to the appearance of the typical EPR spectra of PBN/HER adduct. No PBN/HER spin adduct was detected when DHAE was incubated with 0.1 M PBN in the presence of GSH. In the absence of PBN, DHAE oxidized ascorbic acid to semidehydroascorbyl radical, presumably via an ascorbate-dependent one-electron reduction of HER back to ethanol. Catalase was progressively inactivated by exposure to DHAE-generated HER in a time and HER concentration-dependent manner. Ascorbic acid and PBN gave full protection to catalase against HER-dependent inactivation. The antioxidants 2-tert-butyl-4-methylphenol, propylgallate, and alpha-tocopherol-protected catalase against inactivation by 84, 88, and 39%, respectively. Other antioxidant enzymes were also sensitive to exposure to HER. Glutathione reductase, glutathione peroxidase, and superoxide dismutase were inactivated by 46, 36, and 39%, respectively, by HER. The results reported here plus previous results showing HER interacts with GSH, ascorbate, and alpha-tocopherol suggest that prolonged generation of HER in cells from animals chronically exposed to ethanol may lower the antioxidant defense status, thereby contributing to mechanisms by which ethanol produces a state of oxidative stress and produces toxicity.

Antioxidants↗

PubMed1999

Nitric oxide generation by soybean embryonic axes. Possible effect on mitochondrial function.

Nitric oxide (NO) generation and its effect on mitochondrial enzymes were investigated in soybean embryonic axes at the onset of germination. NO was detected in homogenates from soybean embryonic axes by EPR. Enzymatic sources of NO, such as nitrate reductase activity and nitric oxide synthase, assessed as NADPH-diaphorase activity, were measured in homogenates incubated up to 48 h. Both NO content and the activity of the enzymes showed a similar profile as function of the imbibition time, with maximal levels at 15-24h. Total O2 consumption in enriched-mitochondrial fraction was inhibited by NO in a concentration-dependent manner. O2 consumption dependent on cytochrome oxidase activity was more sensitive than alternative oxidase pathway to NO exposure. Half maximal effects of NO at 0.3 and 3.6 microM were measured for cytochrome oxidase and alternative oxidase, respectively. Enriched-mitochondrial fractions from soybean embryonic axes treated with NO (up to 1 microM) showed increased H2O2 production. The data presented suggest that NO could modulate O2 consumption in soybean embryonic axes. This process could affect the pro-oxidant/antioxidant balance and the cellular energy yield in the germinating embryonic axes, and could have a role in soybean germination.

Electron Spin Resonance Spectroscopy↗

PubMed1999

Derangement of Kupffer cell functioning and hepatotoxicity in hyperthyroid rats subjected to acute iron overload.

Liver oxidative stress, Kupffer cell functioning, and cell injury were studied in control rats and in animals subjected to L-3,3',5-tri-iodothyronine (T3) and/or acute iron overload. Thyroid calorigenesis with increased rates of hepatic O2 uptake was not altered by iron treatment, whereas iron enhanced serum and liver iron levels independently of T3. Liver thiobarbituric acid reactants formation increased by 5.8-, 5.7-, or 11.0-fold by T3, iron, or their combined treatment, respectively. Iron enhanced the content of protein carbonyls independently of T3 administration, whereas glutathione levels decreased in T3- and iron-treated rats (54%) and in T3Fe-treated animals (71%). Colloidal carbon infusion into perfused livers elicited a 109% and 68% increase in O2 uptake in T3 and iron-treated rats over controls. This parameter was decreased (78%) by the joint T3Fe administration and abolished by gadolinium chloride (GdCl3) pretreatment in all experimental groups. Hyperthyroidism and iron overload did not modify the sinusoidal efflux of lactate dehydrogenase, whereas T3Fe-treated rats exhibited a 35-fold increase over control values, with a 54% reduction by GdCl3 pretreatment. Histological studies showed a slight increase in the number or size of Kupffer cells in hyperthyroid rats or in iron overloaded animals, respectively. Kupffer cell hypertrophy and hyperplasia with presence of inflammatory cells and increased hepatic myeloperoxidase activity were found in T3Fe-treated rats. It is concluded that hyperthyroidism increases the susceptibility of the liver to the toxic effects of iron, which seems to be related to the development of a severe oxidative stress status in the tissue, thus contributing to the concomitant liver injury and impairment of Kupffer cell phagocytosis and particle-induced respiratory burst activity.

Animals↗

PubMed1998

Time course study of the influence of acute iron overload on Kupffer cell functioning and hepatotoxicity assessed in the isolated perfused rat liver.

This study tested the hypothesis that acute iron overload (500 mg/kg) alters Kupffer cell functioning by promoting free radical reactions associated with the respiratory burst of liver macrophages, assessed in the isolated perfused rat liver under conditions of Kupffer cell stimulation by carbon infusion and inactivation by gadolinium chloride pretreatment. Total serum and hepatic iron levels were markedly enhanced compared with control values 2 to 24 hours after iron treatment. Total liver O2 uptake progressively increased by iron overload reaching a maximum at 6 hours after treatment, an effect that was completely blocked by GdCl3. Concomitantly, carbon-induced GdCl3-sensitive liver O2 uptake was either enhanced by 119% at 2 hours after iron overload, diminished compared with control values at 4 hours, or abolished at 6 hours. Iron-overloaded rats showed a marked increase in liver sinusoidal lactate dehydrogenase efflux at 4 and 6 hours after treatment, an effect that is exacerbated by carbon infusion and reduced (69%-89%) by GdCl3 pretreatment. Both basal and carbon-induced lactate dehydrogenase effluxes returned to control values at 24 hours after iron overload concomitantly with depression of the basal O2 uptake, without development of iron-induced GdCl3-sensitive respiration or Kupffer cell activation by carbon infusion. It is concluded that iron overload induces a derangement in the Kupffer cell functional status represented by early increases in macrophage-dependent respiratory activity, which may contribute to the concomitant liver injury that developed and to the impairment of both hepatic respiration and the macrophage response to particle stimulation observed at later times after treatment.

Acute Disease↗

PubMed1998

Production of reactive oxygen species by microsomes enriched in specific human cytochrome P450 enzymes.

Few studies have evaluated the production of reactive oxygen intermediates by human microsomes, especially the influence of the specific form of cytochrome P450. Experiments were carried out to evaluate the ability of CYP1A1, 1A2, 2B6, and 3A4 to consume NADPH, reduce iron, and catalyze production of reactive oxygen species. Microsomes enriched in each of these CYPs were obtained from commercial +/- lymphoblast cells that had been transfected with cDNA encoding the specific human CYP. On a per nanomole cytochrome P450 basis, CYP3A4 was the most active P450 evaluated in catalyzing NADPH oxidation, production of superoxide anion radical, NADPH-dependent chemiluminescence, oxidation of dichlorofluorescein diacetate, and reduction of either ferric-EDTA or ferric-citrate. CYP1A1 was the next most reactive CYP, whereas CYP1A2 and 2B6 displayed a comparable, lower activity. Nitric oxide, which reacts with and inactivates hemoproteins, inhibited superoxide production by all the CYPs to a similar extent. Because CYP3A4 is present in high amounts in human liver microsomes and is active in catalyzing the formation of reactive oxygen species, this CYP may make an important contribution in the overall ability of human liver microsomes to generate active oxygen species.

Aryl Hydrocarbon Hydroxylases↗

PubMed1997

Dietary alpha-tocopherol supplementation on antioxidant defenses after in vivo iron overload in rats.

The effect of dietary alpha-tocopherol (alpha-T) supplementation on iron overload-dependent oxidative damage was studied. Male Wistar rats were fed diets supplemented with 2.5% carbonyl iron and/or 200 mg/kg of alpha-tocopheryl acetate for 6 weeks. Oxidation of lipids and proteins were increased by iron overload in rat liver, and alpha-T dietary supplementation effectively prevented these effects. Iron overload decreased both, catalase and Mn-superoxide dismutase activities by 49 and 54%, respectively, with no effect on glutathione peroxidase activity. Alpha-T supplementation did not prevent the inhibition measured in catalase and Mn-superoxide dismutase activities. Iron dietary excess had no effect on liver alpha-T and ubiquinol 9 (UQ9) content. Ubiquinol 10 (UQ10) content after iron overload was decreased by 58 and 54% in whole liver and liver mitochondria, respectively. Alpha-T supplementation led to significant increases in alpha-T, UQ9 and UQ10 content in liver, as compared to control values, and partially prevented the decrease in UQ10 content due to iron excess. The results presented here indicate that initial stages of iron overload led to oxidative damage in liver (evaluated in terms of lipid and protein oxidation) with a decline in antioxidant defenses. alpha-T supplementation affected the liver content of lipid soluble antioxidants, suggesting a concerted antioxidant response at the cellular level to modulate the effect of excess iron availability.

Animals↗

PubMed1997

Inhibition of ferritin-stimulated microsomal production of reactive oxygen intermediates by nitric oxide.

Experiments were carried out to evaluate the effect of nitric oxide exposure on the ability of NADPH-dependent microsomal electron transfer to mobilize iron from ferritin. Such interactions could play a role in potential antioxidant actions of nitric oxide (NO). Preincubation of the microsomes from phenobarbital-treated rats with NO donors such as S-nitroso-D,L-N-acetyl penicillamine (SNAP), S-nitroso-L-glutathione, SIN-1, and DETANONOate followed by centrifugation, washing, and resuspension of the microsomes resulted in a decrease in the ferritin-dependent oxidation of 2',7'-dichlorofluorescein diacetate (DCFDA) or ferritin-catalyzed chemiluminescence compared to microsomes pretreated with buffer. The ferritin-stimulated rate of oxidation of DCFDA or of chemiluminescence was completely restored if the microsomal preincubation with NO donors was performed in the presence of hemoglobin. In contrast to results with ferritin, ferric-stimulated oxidation of the dye was not affected by any of the tested NO donors. The microsomal oxidation of aminopyrine was inhibited after SNAP treatment, indicating that NO inhibited cytochrome P450 catalyzed activity. Inhibition of cytochrome P450 also resulted in an inhibition of microsomal production of superoxide. Similar results were obtained using microsomes from a cloned cell line which express the CYP2E1 isoform. Since superoxide is required for the mobilization of iron from ferritin by microsomes, inhibition of superoxide production as a consequence of NO interaction with cytochrome P450 is likely to be responsible for the prevention of ferritin-catalyzed formation of reactive oxygen species by NO donors. The results suggest that NO could exhibit an antioxidant capacity through its ability of decreasing the activity of iron-heme compounds, such as cytochrome P450, preventing the release of catalytically active iron from ferritin, and thus decreasing the ability to generate oxygen free radicals involved in cytotoxicity.

Animals↗

PubMed1996

Effect of in vivo iron supplementation on oxygen radical production by soybean roots.

Both total iron content and the in vitro rate of iron reduction were higher in roots grown in the presence of exogenously added iron (up to 500 microM) than in roots grown in absence of supplemented iron. In vivo generation of reactive O2 species by intact roots was assessed employing 2',7'-dichlorofluorescein diacetate oxidation to a fluorescent product. Roots developed in the presence of 500 microM Fe-EDTA, showed significantly increased fluorescence (64%), as compared to roots developed in absence of added iron, suggesting that iron supplementation led to oxidative stress in vivo. At the subcellular level, iron content and Fe-EDTA reduction rate were significantly increased in microsomes isolated from roots developed in the presence of exogenously added iron, as compared to microsomes from roots grown in the absence of iron. Microsomes from Fe supplemented plants exhibited a 51% increase in 2',7'-dichlorofluorescein diacetate oxidation rate, a 55% increase in superoxide anion generation, and a four-fold increase in hydroxyl radical production as compared to controls. Iron supplementation did not affect the activity of antioxidant enzymes or the content of total thiols, however alpha-tocopherol content was significantly decreased in the homogenates and the microsomes isolated from roots supplemented with iron, as compared with values in roots developed in absence of iron. These data suggest that in vivo iron supplementation increased oxygen radical generation.

Antioxidants↗

PubMed1996

Role of cytochrome P-450 in the stimulation of microsomal production of reactive oxygen species by ferritin.

Microsomes can remove iron from ferritin by a superoxide-dependent reaction. The released iron can then catalyse formation of a variety of reactive oxygen species. Experiments were carried out to evaluate the role of cytochrome P-450 in the release of iron from ferritin, and whether induction of certain P-450 isoforms alters ferritin-dependent reactive oxygen radical production. Rats were treated with phenobarbital, 3-methylcholanthrene, 4-methylpyrazole, or saline to produce microsomes with varying P-450 content and composition. Oxidation of 2,7'-dichlorofluorescein diacetate to a fluorescent product and chemiluminescence were used as indices of production of reactive oxygen species. The extreme sensitivity of these reactions to trolox, a potent chain-breaking oxidant, indicates the involvement of lipid peroxidation products in these reactions. In the absence of ferritin, formation of reactive oxygen species was higher in microsomes from the treated rats compared to saline controls when results were expressed on a per mg protein basis but not per nmol P-450, suggesting that the increased content of total P-450 (2-fold increases) rather than the population of isoforms was responsible for the increase. Superoxide dismutase had no effect on the non-ferritin catalyzed reactions. Ferritin increased production of reactive oxygen species with all the microsomal preparations; the increase by ferritin was completely prevented by superoxide dismutase. The net increase by ferritin was higher in microsomes from the treated rats compared to saline controls, but this, again, largely reflected the increased content, rather than the type of isoforms of P-450 present. Similar results were obtained with either NADPH or NADH as microsomal reductants, although NADPH was much more effective in supporting ferritin-dependent reactive oxygen formation. In microsomes from phenobarbital-treated rats, anti-CYP2B1/B2 IgG completely prevented the NADPH- and NADH-dependent increases in reactive oxygen formation produced by ferritin. Anti-cytochrome b5 IgG produced partial inhibition of the ferritin-stimulation. These results indicate that P-450, and to a lesser extent, cytochrome b5, play a role in the ferritin-dependent increase in formation of reactive oxygen species with either NADPH or NADH, most likely reflecting the requirement of these enzymes for microsomal production of superoxide anion.

Animals↗

PubMed1995

Role of antioxidants on the erythrocytes resistance to lipid peroxidation after acute iron overload in rats.

Iron overload was developed in rats by ip injection of iron-dextran. Iron concentration in plasma increased 12-fold after 20 h of iron supplementation and unsaturated iron binding capacity (UIBC) drastically decreased in iron overloaded compared to control rats (69 +/- 36 and 177 +/- 19 micrograms/dl, respectively). Lipid peroxidation in plasma increased by 285% and plasma alpha-tocopherol content decreased by 40% after 20 h of iron overload. alpha-Tocopherol supplementation decreased by 30% the measured increase in TBARS content in plasma after iron injection. On the other hand, both iron and TBARS content in erythrocytes were not significantly different in control and iron loaded rats. However, red blood cells from iron treated rats exposed to pro-oxidant conditions showed a significant increase in TBARS content as compared to control erythrocytes. alpha-Tocopherol pretreatment prevented this increase. Moreover, red blood cells from iron loaded rats showed a higher content of TBARS after incubation with plasma from iron-dextran injected rats than after incubation with plasma from control animals. This measured increase was partially prevented by alpha-tocopherol supplementation. Neither the activity of antioxidant enzymes nor the content of alpha-tocopherol in red blood cells were affected by iron overload. Total thiols content was significantly lower (30%) in erythrocytes isolated from iron treated rats. The data presented here suggest that free radical generation catalyzed by metal ions may lead to consumption of thiols. The decrease in thiols content in erythrocytes could afford an appropriate degree of protection and avoid other oxidative damage to these cells.

Animals↗

PubMed1994

Mild iron overload effect on rat liver nuclei.

A single injection of iron-dextran significantly increased iron content in plasma, whole liver, cellular cytosol and liver nuclei. In vitro nuclear rate of Fe(3+)-EDTA reduction was not affected by the treatment. Membrane-bound enzymatic activities in the nuclei were measured after iron overload. Both NADPH- and NADH-dependent cytochrome c reductases were slightly decreased after iron overload, but cytochrome P450 was undetectable after 6 h of iron supplementation. The contents of lipid- and water-soluble antioxidants were measured in isolated nuclei from control and iron-overloaded rats. alpha-Tocopherol and beta-carotene co-elutant were decreased by 40% and 83%, respectively after 6 h of treatment. Nuclear glutathione content was not affected. The rate of generation of superoxide anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical-like species by isolated rat liver nuclei, were decreased by 50%, 40% and 60%, respectively after 6 h of iron supplementation. An identical qualitative response to iron overload was observed with NADPH and NADH. The inactivation of nuclear cytochrome P450, the significant loss in lipid-soluble antioxidants (alpha-tocopherol and beta-carotene) and the decrease in enzyme-dependent oxygen radical generation, suggest that the increase in catalytic active iron induced by iron overload could affect the cellular nuclei functionality.

Animals↗