Dose-dependent effects of chronic iron burden on heart aldehyde and acyloin production in mice

Iron’s chemical structure and its ability to initiate one-electron reactions are properties that cause it to play a major role in the production and metabolism of oxygen free radicals in biological systems. Oxygen free radicals are conjectured to cause cardiac failure in individuals afflicted with disorders of iron overload. We report on the use of both acyloins and aldehydes as markers of oxidative stress in a murine model of chronic iron-overload cardiomyopathy. Twenty mice were randomized to four treatment groups: (1) control (0.2 mL normal saline ip/mouse/d); (2) 100 mg iron (0.05 mL iron dextran/mouse/d); (3) 200 mg iron (0.1 mL iron dexxtran/mouse/d); (4) 400 mg iron (0.2 mL iron dextran/mouse/d). Significant dose-dependent increases in both total heart aldehyde and total heart acyloin concentrations were found. Furthermore, a significant positive correlation existed between the dose of iron administered and each quantified aldehyde and acyloin found in the heart.     

Alteration of free radical metabolism in the brain of mice infected with scrapie agent.

Alteration of free radical metabolism in the mouse brain by scrapie infection was evaluated. The infection of mice with scrapie agent, 87V strain, slightly increased the activities of catalase and glutathione-S-transferase, while it had no effect on glutathione peroxidase, glutathione reductase, and Cu, Zn-superoxide dismutase. Results show that the scrapie infection decreased the activity of mitochondrial Mn-superoxide dismutase by 50% but increased that of monoamine oxidase (p < 0.05). Scrapie infection also increased the rate of mitochondrial superoxide generation (p < 0.05). Following scrapie infection, the level of free-sulfhydryl compounds in brain homogenates slightly decreased, but the content of thiobarbituric-acid-reactive substances and malondialdehyde increased significantly. Electron microscopy indicated that the ultrastructure of mitochondria was destroyed in the brain of scrapie-infected mice. These results suggest that elevated oxygen free radical generation and lowered scavenging activity in mitochondria might cause the free radical damage to the brain. Such deleterious changes in mitochondria may contribute to the development of prion disease.     

Investigations into the systemic production of aldehyde-derived peroxidation products in a murine model of acute iron poisoning: a dose response study

Acute iron poisoning remains a leading cause of morbidity and mortality in pre-school aged children in North America. Acute iron poisoning leads to organ damage, such as respiratory difficulties, cardiac arrhythmias, and possible death. The mechanism of iron toxicity is not fully understood, though it is thought that free iron is able to catalyze the production of harmful oxygen free radicals, which can damage all biochemical classes including lipid membranes, proteins, and DNA. Accordingly, we hypothesized that acute iron loading results in dose-dependent increases in oxygen free radical production, as quantified by the cytotoxic aldehydes hexanal, 4-hydroxynonenal, and malondialdehyde, in an experimental murine model. In support of our hypothesis, significant dose-dependent increases in all aldehydes investigated were reported in comparison to controls (p < 0.001). This murine model will assist in providing a better understanding of possible mechanism(s) of injury and organ dysfunction following acute iron poisoning, and for the development and evaluation of treatment regimes.     

The role of cellular oxidases and catalytic iron in the pathogenesis of ethanol-induced liver injury

Free radical generation and catalytic iron have been implicated in the pathogenesis of alcohol-induced liver injury but the source of free radicals is a subject of controversy. The mechanism of ethanol-induced liver injury was investigated in isolated hepatocytes from a rodent model of iron loading in which free radical generation was measured by the determination of alkane production (ethane and pentane). Iron loading (125mg/kg i.p.) increased hepatic non-heme iron 3-fold, increased the prooxidant activity of cytosolic ultrafiltrates 2-fold and doubled ethanol-induced alkane production. The addition of desferrioxamine (20μM), a tight chelator of iron, completely abolished alkane production indicating the importance of catalytic iron. The role of cellular oxidases as a source of ethanol induced free radicals was studied through the use of selective inhibitors. In both the presence and absence of iron loading, selective inhibition of xanthine oxidase with oxipurinol(20μM) diminished ethanol-induced alkane production 0–40%, inhibition of aldehyde oxidase with menadione (20μM) diminished alkane production 36–75%, while the inhibition of aldehyde and xanthine oxidase by feeding tungstate (100mg/kg/day) virtually abolished alkane production. Addition of acetaldehyde(50μM) to hepatocytes generated alkanes at rates comparable to those achieved with ethanol indicating the importance of acetaldehyde metabolism in free radical generation. The cellular oxidases (aldehyde and xanthine oxidase) along with catalytic iron play a fundamental role in the pathogenesis of free radical injury due to ethanol.     

Free radical activity, antioxidant enzyme, and glutathione changes with muscle stretch injury in rabbits.

The present study investigated changes in rate of free radical production, antioxidant enzyme activity, and glutathione status immediately after and 24 h after acute muscle stretch injury in 18 male New Zealand White rabbits. There was no change in free radical production in injured muscles, compared with noninjured controls, immediately after injury (time 0; P = 0.782). However, at 24 h postinjury, there was a 25% increase in free radical production in the injured muscles. Overall, there was an interaction (time and treatment) effect (P = 0.005) for free radical production. Antioxidant enzyme activity demonstrated a treatment (injured vs. control) and interaction effect for both glutathione peroxidase (P = 0.015) and glutathione reductase (P = 0.041). There was no evidence of lipid peroxidation damage, as measured by muscle malondialdehyde content. An interaction effect occurred for both reduced glutathione (P = 0.008) and total glutathione (P = 0.015). Morphological analysis (hematoxylin and eosin staining) showed significant polymorphonuclear cell infiltration of the damaged region at 24 h postinjury. We conclude that acute mechanical muscle stretch injury results in increased free radical production within 24 h after injury. Antioxidant enzyme and glutathione systems also appear to be affected during this early postinjury period.     

Comparison of injectable iron complexes in their ability to iron load tissues and to induce oxidative stress

Iron and copper homeostasis have been studied in various tissues after iron-loading with the polynuclear ferric hydroxide carbohydrate complexes, iron dextran, iron polymaltose, iron sucrose and iron gluconate for four weeks. There were significant increases in the iron content of the different rat tissues compared to controls, with the exception of the brain, which showed no change in its iron content following iron loading. However, the level of iron loading in the different tissues varied according to the preparation administered and only iron dextran was able to significantly increase the iron content of both broncho-alveolar macrophages and heart. The hepatic copper content decreased with iron loading, although this did not reach significance. However the copper content did not alter in the iron loaded broncho-alveolar macrophages. Despite such increases in hepatic iron content, there was little evidence of changes in oxidative stress, the activities of cytosolic (apart from iron dextran) or mitochondrial hepatic superoxide dismutase, SOD, were similar to that of the control rats, confirming the fact that the low reduction potential of these compounds prevents the reduction of the ferric moiety. It was not necessary for macrophages to significantly increase their iron content to initiate changes in NO^. release. Iron gluconate and iron sucrose increased NO^. release, while iron polymaltose and iron dextran decreased NO^. release although only the latter iron preparation significantly increased their iron content. It may be that the speciation of iron within the macrophage is an important determinant in changes in NO^. release after ex vivo stimulation. We conclude that tissues loaded with iron by such polynuclear iron complexes have variable loading despite the comparable iron dose. However, there was little evidence for participation of the accumulated iron in free radical reactions although there was some evidence for alteration in immune function of broncho-alveolar macrophages.     

A re-evaluation of the antioxidant activity of purified carnosine

The antioxidant activity of carnosine has been re-evaluated due to the presence of contaminating hydrazine in commercial carnosine preparations. Purified carnosine is capable of scavenging peroxyl radicals. Inhibition of the oxidation of phosphatidylcholine liposomes by purified carnosine is greater in the presence of copper than iron, a phenomenon likely to be due to the copper chelating properties of carnosine. Purified carnosine is capable of forming adducts with aldehydic lipid oxidation products. Adduct formation is greatest for alpha,beta-monounsaturated followed by polyunsaturated and saturated aldehydes. While the ability of carnosine to form adducts with aldehydic lipid oxidation products is lower than other compounds such as glutathione, the higher concentrations of carnosine in skeletal muscle are likely to make it the most important molecule that forms aldehyde adducts. Monitoring changes in carnosine concentrations in oxidizing skeletal muscle shows that carnosine oxidation does not occur until the later stages of oxidation suggesting that carnosine may not be as effective free radical scavenger in vivo as other antioxidants like alpha-tocopherol.     

Dietary saturated or polyunsaturated fat and copper deficiency in the rat

Dietary fat-type and copper (Cu) deficiency have been independently identified as potentially important factors in the etiology of ischemic heart disease (IHD); a disease that has been linked to inflammation and oxygen free radical (OFR) mediated damage. Group (n = 6) of male, weanling, Wistar rats were provided ad libitum with deionized water and control or low Cu diets containing (200 g/kg) either saturated or polyunsaturated fatty acids (SFA or PUFA, respectively) for 56 d. Measurement of several indices of Cu status indicated that both groups fed the low Cu diets were Cu-deficient. SFA consumption resulted in significantly increased hepatic Cu (p less than 0.001) and iron (Fe) (p less than 0.001) concentrations and xanthine oxidase activity (p less than 0.05) and significantly decreased hepatic glucose-6-phosphate dehydrogenase activity (p less than 0.001). Although Cu deficiency resulted in significantly decreased hepatic copper-zinc superoxide dismutase (CuZnSOD) activity (p less than 0.01), no significant effect on the activities of the other hepatic antioxidant enzymes, manganese superoxide dismutase, catalase, and glutathione peroxidase, or glutathione reductase, were observed. Cu deficiency also resulted in significantly decreased hepatic Cu levels (p less than 0.001) and cytochrome c oxidase activity (p less than 0.01). No significant difference in hepatic thiobarbituric acid reactive substances (TBARS), a measure of lipid peroxidation, was found between groups consuming SFA or PUFA, but both Cu-deficient groups exhibited significantly increased hepatic TBARS (p less than 0.001), compared to controls. This was probably owing to the significantly decreased hepatic CuZnSOD activity observed in the Cu-deficient, compared to control animals.     

Selenium compounds have disparate abilities to impose oxidative stress and induce apoptosis

The cancer chemopreventive effect of selenium cannot be fully accounted for by the role of selenium as a component of the antioxidant enzyme glutathione peroxidase, which suggests that chemoprevention occurs by another mechanism. Several studies have shown that thiol oxidation and free radical generation occur as a consequence of selenium catalysis and toxicity. In the present study, we evaluated three different selenium compounds; selenite, selenocystamine, and selenomethionine to determine the relative importance of the prooxidative effects of these compounds with regard to their ability to induce apoptosis. The experimental results suggest that, in addition to supporting an increased activity of glutathione peroxidase, an antioxidant function that the three selenium compounds did with equal efficacy, catalytic selenite, and selenocystamine generated 8-hydroxydeoxyguanosine DNA adducts, induced apoptosis and were found to be cytotoxic in mouse keratinocytes. The noncatalytic selenomethionine was not cytotoxic, did not generate 8-hydroxydeoxyguanosine adducts and did not induce cellular apoptosis at any of the selenium concentrations studied. In keratinocytes, apoptosis may be initiated by superoxide (O₂^•−) and oxidative free radicals that are generated by selenite and selenocystamine, but not by selenomethionine.     

Age-dependent peculiarities modulation of activity of aldehyde scavenger enzymes in mitochondria of rat thigh muscle during stress

The purpose of this study was a comparative investigation of activity of aldehyde scavenger enzymes in mitochondrial fraction of a thigh muscle in intact and immobilized rats of different ages. It has been shown that 12-month-old (adult) rats have high basal levels of aldehyde dehydrogenase, aldehyde reductase and glutathione transferase activity in mitochondrial fraction of thigh muscle. Aldehyde dehydrogenase activity increases during immobilization stress in adult rats. This change promote to enhance the effectiveness of utilization of carbonyl products of free radical oxidation in mitochondria of skeletal muscle of 12-month-old rats during stress. Immobilization of old and pubertal rats is accompanied by metabolic preconditions leading to accumulation of endogenous aldehydes in mitochondria, and, as a result, to the injury of muscular fibers and intensification of sarcopenia manifestations.     

Acidosis Potentiates Oxidative Neuronal Death by Multiple Mechanisms

Both acidosis and oxidative stress contribute to ischemic brain injury. The present study examines interactions between acidosis and oxidative stress in murine cortical cultures. Acidosis (pH 6.2) was found to potentiate markedly neuronal death induced by H2O2 exposure. To determine if this effect was mediated by decreased antioxidant capacity at low pH, the activities of several antioxidant enzymes were measured. Acidosis was found to reduce the activities of glutathione peroxidase and glutathione S-transferase by 50-60% (p < 0.001) and the activity of glutathione reductase by 20% (p < 0.01) in lysates of the cortical cultures. Like acidosis, direct inhibition of glutathione peroxidase with mercaptosuccinate also potentiated H2O2 toxicity. Because acidosis may accelerate hydroxyl radical production by the Fenton reaction, the effect of iron chelators was also examined. Both desferrioxamine and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, two structurally different iron chelators, significantly reduced H2O2-induced neuronal death under both pH 7.2 and pH 6.2 conditions. These results suggest that the increased cell death produced by severe acidosis during cerebral ischemia may result in part from exacerbation of oxidative injury. This exacerbation may result from both impaired antioxidant enzyme functions and increased intracellular free iron levels.     

Lipid peroxidation in the liver of carcinogen-resistant rats

Recently, we developed a new strain of rats that exhibit marked resistance to the hepatotoxic and carcinogenic actions of 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) and some other carcinogens. In this work, we compared lipid peroxidation in the liver of these carcinogen-resistant (R) rats and the parental Donryu strain rats that are sensitive (S) to hazardous actions of these carcinogens. The liver microsomal fractions of the R group contained less amounts of polyunsaturated fatty acids. Microsomal lipid peroxidation in the presence of exogenous NADPH was much lower in R rats than in S rats. Liver microsomes of R rats were much less active than those of S rats also in producing 4-hydroxynonenal, carbonyl compounds and conjugated diene. The hepatic contents of ascorbic acid, glutathione, alpha-tocopherol and coenzyme Q in the R rats were similar to those in S rats. The activities of the free radical scavenger enzymes, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), in the two groups were also similar. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are both thought to function in disposal of these cytotoxic aldehydes. The liver microsomal and mitochondrial ALDH activities of the two groups were similar. The ADH activity of the liver cytosolic fraction of R rats was nearly twice that of S rats, as measured with 4-hydroxynonenal as substrate. The higher ADH activity may explain the decreased lipid peroxidation in R rats at least partly, if this enzyme is involved in lipid peroxidation.     

芘对苦草的生物毒性效应

通过静态模拟实验,研究了不同浓度(0.01、0.02、0.05、0.07和0.1 mg·L^-1)芘暴露10 d后,芘在苦草中的富集,以及芘对苦草茎叶中自由基含量、抗氧化系统以及叶绿素、可溶性糖等的影响.结果表明: 芘能够大量富集在苦草叶部;自由基信号强度、过氧化物酶(POD)活性和脂质过氧化产物丙二醛(MDA)含量持续升高;当污染加重时,这种升高现象有减弱的趋势,且三者有较好的相关性;谷胱甘肽S转移酶(GST)活性和氧化型谷胱甘肽(GSSG)含量持续增加,而还原型谷胱甘肽(GSH)含量持续降低;随着芘浓度的增加,叶绿素含量降低,而可溶性糖含量升高.苦草对芘暴露比较敏感,0.01 mg·L^-1即已显示胁迫效应.     

Nickel-mediated inhibition in the glutathione-dependent protection against lipid peroxidation

Glutathione protects liver microsomes against the rapid onset of lipid peroxidation via a sulfhydryl dependent heat labile factor known as free radical reductase. The administration of nickel to mice resulted in an inhibition in the activity of free radical reductase, and enhanced lipid peroxidation and the activity of glutathione S-transferase in a dose dependent manner. The pretreatment of cyclam, a known specific chelator of nickel restored free radical reductase and glutathione S-transferase activities and alleviated nickel mediated enhancement of lipid peroxidation. Our results indicate that nickel-mediated inhibition in free radical reductase activity and activation of glutathione S-transferase may be due to the interaction of nickel with sensitive-SH groups located on these proteins.     

Thiol and Mn(2+)-mediated oxidation of veratryl alcohol by horseradish peroxidase.

Horseradish peroxidase has been shown to catalyze the oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) and benzyl alcohol to the respective aldehydes in the presence of reduced glutathione, MnCl2, and an organic acid metal chelator such as lactate. The oxidation is most likely the result of hydrogen abstraction from the benzylic carbon of the substrate alcohol leading to eventual disproportionation to the aldehyde product. An aromatic cation radical intermediate, as would be formed during the oxidation of veratryl alcohol in the lignin peroxidase-H2O2 system, is not formed during the horseradish peroxidase-catalyzed reaction. In addition to glutathione, dithiothreitol, L-cysteine, and beta-mercaptoethanol are capable of promoting veratryl alcohol oxidation. Non-thiol reductants, such as ascorbate or dihydroxyfumarate (known substrates of horseradish peroxidase), do not support oxidation of veratryl alcohol. Spectral evidence indicates that horseradish peroxidase compound II is formed during the oxidation reaction. Furthermore, electron spin resonance studies indicate that glutathione is oxidized to the thiyl radical. However, in the absence of Mn2+, the thiyl radical is unable to promote the oxidation of veratryl alcohol. In addition, Mn3+ is unable to promote the oxidation of veratryl alcohol in the absence of glutathione. These results suggest that the ultimate oxidant of veratryl alcohol is a Mn(3+)-GSH or Mn(2+)-GS. complex (where GS. is the glutathiyl radical).     

The dose-dependent effect of 3,5-dicarbomethoxyphenylbiguanide on activity of the glutathione antioxidant system in heart and blood serum of rats with experimental myocardial infarction

Administration of a synthetic compound with predicted anti-ischemic and cardioprotective activity, 3,5-dicarbomethoxyphenylbiguanide (3,5-DCMPBG) to rats with experimental myocardial infarction led to a decrease in the lipid peroxidation level, glutathione peroxidase activity, the level of reduced glutathione, activity of NADP-isocitrate dehydrogenase in the heart and blood serum, and activity of glucoso-6-phosphate dehydrogenase in heart in comparison with their levels in untreated animals with myocardial infarction. This may be attributed to a decrease of free radical processes and reduction of antioxidant system loading induced by the protective effect of the administered compound. At the same time the increase glutathione reductase activity observed under these conditions in the heart and blood serum is probably associated with specific influence of 3,5-DCMPBG on this enzyme.     

Melatonin controls oxidative stress and modulates iron, ferritin, and transferrin levels in adriamycin treated rats

AIM: Chemotherapy with adriamycin (ADR) is limited by its iron-mediated pro-oxidant toxicity. Because melatonin (MLT) is a broad spectrum antioxidant, we investigated the ability of MLT to control iron, its binding proteins, and the oxidative damage induced by ADR. MAIN METHODS: ADR was given as single i.p. dose of 10 mg kg(-1) body weight into male rats. MLT at a dose of 15 mg kg(-1) was injected daily for 5 days before ADR treatment followed by another injection for 5 days. Biochemical methods were used for this investigation. KEY FINDINGS: ADR injection caused elevations in plasma creatine kinase isoenzyme, lactic dehydrogenase, and aminotransferases, iron, ferritin, and transferrin. These changes were associated with increases in lipid peroxidation and protein oxidation as well as decreases in glutathione (GSH) levels and glutathione-S-transferase (GST) activity, while glutathione peroxidase (GSH-Px), and catalase (CAT) activity were elevated in the heart and liver of ADR treated rats. In the MLT+ADR group, the cardiac and hepatic function parameters and the levels of iron, transferrin and ferritin in plasma were normalized to control levels. The rats that were subjected to MLT+ADR had normalized CAT and GSH-Px activity and decreased TBARS and protein carbonyl levels compared the group only treated with ADR. GST activity and GSH concentration in the heart and liver were normalized when MLT accompanied ADR treatment. SIGNIFICANCE: MLT ameliorated oxidative stress by controlling iron, and binding protein levels in ADR treated rats demonstrating the usefulness of adriamycin in cancer chemotherapy and allowing a better management of iron levels.     

The cytotoxic activity of lactoperoxidase: enhancement and inhibition by neuroactive compounds

Neuronal death associated with Parkinson's disease is commonly believed to be caused by oxygen- and nitrogen-derived free radical species. Some years ago, however, we showed that peroxidase from the midbrain of dogs is able to kill various cell types, including neuroblastoma cells (M. B. Grisham et al., J. Neurochem. 48: 876-882: 1987). We postulated that a nigral peroxidase may play a significant role in the degeneration of dopaminergic neurons in Parkinson's disease. To further establish proof of principle, we recently performed a series of experiments using horseradish peroxidase and lactoperoxidase. We showed that the cytotoxic activity of lactoperoxidase is fully inhibited by physiological concentrations of dopamine, reduced glutathione, and L-cysteine, as well as by micromolar concentrations of apomorphine, desferal, aspirin, and uric acid. l-Methyl-4-phenyl-1,2-dihydropyridine (MPDP) and l-methyl-4-phenylpyridinium (MPP+) augment the cytotoxic activity, whereas l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, deprenyl, and pargyline had minimal or no effect. We also showed that horseradish peroxidase catalyzes the oxidation of MPDP to MPP+. Thus, contrary to the generally accepted theory that the in vivo oxidation of MPDP occurs spontaneously, this reaction may be catalyzed by a brain peroxidase. These observations lend further support to the suggestion that a brain peroxidase may play an important role in the metabolic events associated with Parkinson's disease.     

Free radical scavenging activity of Cleome gynandra L. leaves on adjuvant induced arthritis in rats

The generation of free radicals has been implicated in the causation of several diseases of known and unknown etiologies such as, rheumatoid arthritis, diabetes, cancer, etc., and compounds that can scavenge free radicals have great potential in ameliorating these disease processes. The present study was aimed to investigate the possible anti-oxidant potential of Cleome gynandra leaf extract at a dose of 150 mg/kg body weight for 30 days on adjuvant induced arthritis in experimental rats. Oral administration of C. gynandra leaf extract significantly increased the levels of lipid peroxides and activities of catalase, glutathione peroxidase and decreased the levels of reduced glutathione and superoxide dismutase activity in arthritis induced rats. The free radical scavenging activity of the plant was further evidenced by histological observations made on the limb tissue. The presence of biologically active ingredients and vital trace elements in the leaves readily account for free radical scavenging property of C. gynandra. (Mol Cell Biochem 276: 71–80, 2005)     

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.