Lipopolysaccharide (LPS) induction of nitric oxide synthase-2 and cyclooxygenase-2 is impaired in fructose overloaded rats.

AIMS: Fructose (F) overload in rats induces metabolic dysfunctions that resemble the human metabolic syndrome. In this paper, we aimed to investigate the response of F overload rats to lipopolysaccharide (LPS) challenge in terms of nitric oxide (NO) production and prostanoids (PR) release. MAIN METHODS: NO blood steady-state concentration was monitored through the detection of nitrosyl-hemoglobin complexes (NO-Hb) by electronic spin resonance. Production of 6-keto PGF(1)α, PGE(2), PGF(2)α and TXB(2) was measured in aorta and mesenteric beds by HPLC. Western blot analysis was used to examine the changes in the expression levels of NOS-2 and COX-2 in aorta. KEY FINDINGS: Our results showed that increases in NO circulating steady-state concentration and PR production by aorta and mesenteric beds 6h after LPS administration were significantly attenuated in F overload rats with respect to control animals. Oxidative stress parameters were equally affected in the presence or absence of the F treatment. Aorta protein levels of NOS-2 and COX-2, two enzymes inducible by LPS, were significantly lower in F overload rats with respect to control rats at the end of the treatment (-39% and -61% for NOS-2 and COX-2 respectively). SIGNIFICANCE: These results suggest that the metabolic alterations established by 15 weeks of F overload should affect the response to LPS challenge due to an attenuation in the induction of NOS-2 and COX-2. This effect would be one of the components contributing to abnormalities in the course of the inflammatory response in other conditions associated to insulin resistance, such as diabetes.

Forms of iron binding in the cells and the chemical features of chelation therapy.

Iron is essential for human life, however it can be toxic through Fe ability to generate oxygen-derived radicals. This work reviews the main features of Fe binding in the cell and its association to these processes. The chemical nature of the Fe extracted by chelation therapy in pathophysiological situations is also analyzed.

Effects of seasonal and latitudinal cold on oxidative stress parameters and activation of hypoxia inducible factor (HIF-1) in zoarcid fish.

Acute, short term cooling of North Sea eelpout Zoarces viviparus is associated with a reduction of tissue redox state and activation of hypoxia inducible factor (HIF-1) in the liver. The present study explores the response of HIF-1 to seasonal cold in Zoarces viviparus, and to latitudinal cold by comparing the eurythermal North Sea fish to stenothermal Antarctic eelpout (Pachycara brachycephalum). Hypoxic signalling (HIF-1 DNA binding activity) was studied in liver of summer and winter North Sea eelpout as well as of Antarctic eelpout at habitat temperature of 0 degrees C and after long-term warming to 5 degrees C. Biochemical parameters like tissue iron content, glutathione redox ratio, and oxidative stress indicators were analyzed to see whether the cellular redox state or reactive oxygen species formation and HIF activation in the fish correlate. HIF-1 DNA binding activity was significantly higher at cold temperature, both in the interspecific comparison, polar vs. temperate species, and when comparing winter and summer North Sea eelpout. Compared at the low acclimation temperatures (0 degrees C for the polar and 6 degrees C for the temperate eelpout) the polar fish showed lower levels of lipid peroxidation although the liver microsomal fraction turned out to be more susceptible to lipid radical formation. The level of radical scavenger, glutathione, was twofold higher in polar than in North Sea eelpout and also oxidised to over 50%. Under both conditions of cold exposure, latitudinal cold in the Antarctic and seasonal cold in the North Sea eelpout, the glutathione redox ratio was more oxidised when compared to the warmer condition. However, oxidative damage parameters (protein carbonyls and thiobarbituric acid reactive substances (TBARS) were elevated only during seasonal cold exposure in Z. viviparus. Obviously, Antarctic eelpout are keeping oxidative defence mechanisms high enough to avoid accumulation of oxidative damage products at low habitat temperature. The paper discusses how HIF could be instrumental in cold adaptation in fish.

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.

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.

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.

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.

Cytotoxicity and apoptosis produced by arachidonic acid in HepG2 cells overexpressing human cytochrome P-4502E1.

The goal of the current study was to evaluate the effects of arachidonic acid, as a representative polyunsaturated fatty acid, on the viability of a HepG2 cell line, which has been transduced to express human cytochrome P4502E1 (CYP2E1). Arachidonic acid produced a concentration- and time-dependent toxicity to HepG2-MV2E1-9 cells, which express CYP2E1, but little or no toxicity was found with control cells. In contrast to arachidonic acid, oleic acid was not toxic to the HepG2-MV2E1-9 cells. The cytotoxicity of arachidonic acid involved a lipid peroxidation type of mechanism since toxicity was enhanced after depletion of cellular glutathione; formation of malondialdehyde and 4-hydroxy-2-nonenal was markedly elevated in the cells expressing CYP2E1, and toxicity was prevented by antioxidants and the iron chelator desferrioxamine. The CYP2E1-dependent arachidonic acid toxicity appeared to involve apoptosis, as demonstrated by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and DNA laddering experiments. Trolox, which prevented toxicity of arachidonic acid, also prevented the apoptosis. Transfection with a plasmid containing bcl-2 resulted in complete protection against the CYP2E1-dependent arachidonic acid toxicity. It is proposed that elevated production of reactive oxygen intermediates by cells expressing CYP2E1 can cause lipid peroxidation, which subsequently promotes apoptosis and cell toxicity when the cells are enriched with polyunsaturated fatty acids such as arachidonic acid.

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.

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.

Cytotoxicity and apoptosis produced by arachidonic acid in Hep G2 cells overexpressing human cytochrome P4502E1.

The goal of the current study was to evaluate the effects of arachidonic acid, as a representative polyunsaturated fatty acid, on the viability of a Hep G2 cell line, which has been transduced to express human cytochrome P4502E1 (CYP2E1). Arachidonic acid produced a concentration- and time-dependent toxicity to Hep G2-MV2E1-9 cells, which express CYP2E1, but little or no toxicity was found with control Hep G2-MV-5 cells, which were infected with retrovirus lacking human CYP2E1 cDNA. In contrast to arachidonic acid, oleic acid was not toxic to the Hep G2-MV2E1-9 cells. The cytotoxicity of arachidonic acid appeared to involve a lipid peroxidation type of mechanism since toxicity was enhanced after depletion of cellular glutathione; formation of malondialdehyde and 4-hydroxy-2-nonenal was markedly elevated in the cells expressing CYP2E1, and toxicity was prevented by antioxidants such as alpha-tocopherol phosphate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), propylgallate, ascorbate, and diphenylphenylenediamine, and the iron chelator desferrioxamine. Transfection of the Hep G2-MV2E1-9 cells with plasmid containing CYP2E1 in the sense orientation enhanced the arachidonic acid toxicity, whereas transfection with plasmid containing CYP2E1 in the antisense orientation decreased toxicity. The CYP2E1-dependent arachidonic acid toxicity appeared to involve apoptosis, as demonstrated by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and DNA laddering experiments. Trolox, which prevented toxicity of arachidonic acid, also prevented the apoptosis. Transfection with a plasmid containing bcl-2 resulted in complete protection against the CYP2E1-dependent arachidonic acid toxicity. It is proposed that elevated production of reactive oxygen intermediates by cells expressing CYP2E1 can cause lipid peroxidation, which subsequently promotes apoptosis and cell toxicity when the cells are enriched with polyunsaturated fatty acids such as arachidonic acid. The Hep G2-MV2E1-9 cells appear to be a valuable model to study interaction between CYP2E1, polyunsaturated fatty acids, reactive radicals, and the consequence of these interactions on cell viability and to reproduce several of the key features associated with ethanol hepatotoxicity in the intragastric infusion model of ethanol treatment.

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.

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.

Effect of mild iron overload on liver and kidney lipid peroxidation.

1. Hepatotoxicity is the most common finding in patients with iron overload since the liver is the major recipient of iron excess, even though the kidney could be a target of iron toxicity. The effect of iron overload was studied in the early stages after iron-dextran injection in rats, as a model for secondary hemocromatosis. 2. Total hepatic and kidney iron content was markedly elevated over control values 20 h after the iron administration. Plasma GOT, GPT and LDH activities were not affected, suggesting that liver cell permeability was not affected by necrosis. 3. Spontaneous liver chemiluminescence was measured as an indicator of oxidative stress and lipid peroxidation. Light emission was increased four-fold 6 h after iron supplementation. 4. Increases in the generation of thiobarbituric acid reactive substances (TBARS in liver and kidney homogenates were detected after iron administration. 5. The activities of catalase, SOD and glutathione peroxidase were determined. Enzymatic activities declined in liver homogenates by 25, 36 and 32%, respectively, 20 h after iron injection. These activities were not affected in kidney as compared to control values, except for SOD activity that was decreased by 26%. 6. The content of alpha-tocopherol was decreased by 31% in whole kidney homogenates and by 40% in plasma. 7. Our data indicate that lipid peroxidation occurs after mild iron overload both in liver and kidney. Enzymatic antioxidants are consumed significantly in liver and alpha-tocopherol content decreases in kidney, suggesting an organ-specific antioxidant effect.

Effect of mild iron overload on liver and kidney lipid peroxidation

1. Hepatotoxicity is the most common finding in patients with iron overload since the liver is the major recipient of iron excess, even though the kidney could be a target of iron toxicity. The effect of iron overload was studied in the early stages after iron-dextran injection inrats, as a model for secondary hemocromatosis. 2. Total hepatic and kidney iron content was markedly over control values 20h after the iron administration. Plasma GOT, GPT and LDH activities were not affected, suggesting that liver cell permeability was not affected by necrosis. 3. Spontaneous liver chemiluminescence was measured as an indicator of oxidative stress and lipid peroxidation. Light emission was increased four-fold 6 h after iron supplementation. 4. Increases in the generation of thiobarbituric acid reactive substances (TBARS) in liver and kidney homogenates were detected after iron administration. 5. The activities of catalase, SOD and glutathione peroxidase were determined. Enzymatic activities declined in liver homogenates by 25, 36 and 32 por cento, respectively, 20 h after iron injection. These activities were not affected in kidney as compared to control values, except for SOD activity that was decreased by 26 por cento. 6. The content of alfa-tocopherol was decreased by 31 por cento in whole kidney homogenates and by 40 por cento in plasma. 7. Our data indicate that lipid peroxidation occurs after mild iron overload both in liver and kidney. Enzymatic antioxidantes are consumed significantly in liver and alfa-tocopherol content decreases in kidney, suggesting an organ-specific antioxidant effect

Resistance of rat kidney mitochondrial membranes to oxidation induced by acute iron overload.

The effect of iron-overload on rat kidney was studied after a single injection of iron-dextran. Total iron content in kidney and isolated kidney mitochondria was markedly elevated over control values. To assess mitochondrial damage by iron overload, succinate-cytochrome c reductase and NADH-cytochrome c reductase activities as well as the rate of succinate-dependent hydrogen peroxide generation were measured. None of these activities were significantly affected by acute iron overload. The net content and the rate of TBARS (thiobarbituric acid reactive species) formation in kidney homogenates from iron-treated rats was significantly higher than that of control animals. Total superoxide dismutase activity in the homogenates from iron overloaded kidney was decreased by 26%, as compared to controls. Catalase, glutathione peroxidase, and Mn-superoxide dismutase activities were not affected by the treatment. The content of alpha-tocopherol was consistently decreased in whole kidney homogenates (-31%), mitochondria from kidney medulla (-31%) and cortex (-34%), from iron-overloaded rats. Our data suggest that iron dextran treatment does not affect kidney integrity, even though increases in lipid peroxidation occur. Vitamin E appears to be effective in controlling iron-dextran dependent radical generation in kidney.

Hepatic chemiluminescence and lipid peroxidation in mild iron overload.

The effect of iron-overload on both hepatic lipid peroxidation and chemiluminescence was studied in early stages after iron-dextran injection. Total hepatic iron content was markedly elevated over control values 2-6 h after iron dose. A 4-fold increase in light emission was detected after 4-6 h after iron injection. Plasma GOT, GPT and LDH activities were not affected by the treatment suggesting that cell permeability was not affected by necrosis. Increases in the generation of thiobarbituric acid reactive substances (TBARS) and chemiluminescence in liver homogenates, were determined as a function of time after iron administration, in the presence of NADPH as cofactor. Under the same experimental conditions, microsomal cytochrome P-450 content was decreased by 40%, 2 h after iron treatment. To evaluate liver antioxidant defenses, catalase, superoxide dismutase and glutathione peroxidase activities were determined. Glutathione peroxidase activity in the homogenate was not affected by the treatment. Catalase and superoxide dismutase activities declined by 25 and 36%, respectively, compared with control values 4 h after the iron dose. Our data suggest that lipid peroxidation occurs after mild iron overload even though the liver remains functional.

Superoxide anion and hydrogen peroxide metabolism in soybean embryonic axes during germination.

The total rate of mitochondrial O2- production in the presence of NADH as substrate increased from 200 to 1340 pmol/min per axis between 2 and 30 h of imbibition. The activities of the enzymes involved in hydroperoxide metabolism, e.g., superoxide dismutase, catalase, peroxidase and glutathione and ascorbate peroxidases, markedly changed during the germination of soybean embryonic axes. Superoxide dismutase was the enzymatic activity affected the most during the initial stages of germination. Intracellular O2- steady-state concentration, calculated from the rate of O2- production and superoxide dismutase activity, showed a 2-fold increase from 2 x 10(-8) M to 4 x 10(-8) M in germination phase I, declined in phase II to 2 x 10(-8) M and remained constant over the rest of the incubation period. The reaction of H2O2 and luminol catalyzed by Co2+ was utilized to measure H2O2 diffused out of the soybean axes after 5 to 10 min of incubation. The catalase-sensitive luminol emission of diffusates prepared from axes previously imbibed from 2 to 30 h corresponded to a H2O2 intracellular steady-state concentration in the range of 0.3 to 0.9 microM. The activity of metal-containing antioxidant enzymes was determined in the extracellular fluid. Cell wall peroxidase activity increased from 10 to 300 mumol/min per mg protein and appears as a potentially important pathway for H2O2 utilization. Hydrogen peroxide metabolism in soybean embryonic axes during early inhibition appears to have the following main features: (a) mitochondrial membranes are the most important source of cytosolic O2- and H2O2; (b) H2O2 is regulated at a steady-state concentration of 0.3-0.9 microM; (c) catalase is the main enzyme in terms of H2O2 utilization; (d) H2O2 exo-diffusion is quantitatively important destiny of intracellular H2O2; and (e) extracellular peroxidase located at the cell wall affords an enzymatic system able to use diffused H2O2.

Adriamycin effects on hydroperoxide metabolism and growth of human breast tumor cells.

Human breast tumor cells MCF-7 were grown during 5 days in the presence of Adriamycin and the IC50 was 50 nM with the highest sublethal concentration 0.1 microM. At this latter concentration Adriamycin produced a complete inhibition of cell division and a partial reversion to a normal breast epithelial appearance. Similar effects of Adriamycin were observed in cells cultured in the presence of 10% FBS and in a chemically defined medium, with Se-glutathione peroxidase activities of 3.8 and 1.3 U/mg of protein, respectively. Cell size and cell oxygen uptake were increased by 41% and by 50%, respectively, in Adriamycin-treated cells. The spontaneous chemiluminescence of monolayers of intact MCF-7 cells (81 +/- 9 cps/mg protein) was increased by 48% in the Adriamycin-treated cultures (120 +/- 11 cps/mg of protein) in agreement with a 91% higher concentration of malondialdehyde in the same cultures. Adriamycin treatment produced a 71% increase in the steady state concentration of H2O2, which was estimated assuming diffusion equilibrium with the external medium, from 1.38 microM in the control cells to 2.38 microM in the treated cells. Cyanide-insensitive respiration was also higher in the cells exposed to the drug than in the control cells. Adriamycin did not affect the activity of the antioxidant enzymes, Cu-Zn and Mn-superoxide dismutase, Se and non-Se-glutathione peroxidase, and catalase. These results contribute to the current hypothesis that oxygen free radicals produced by Adriamycin redox cycling are responsible for at least part of the cytotoxic effects due to this drug.