Iron content and degree of lipid peroxidation in liver mitochondria isolated from iron-loaded rats

1.The content of non-heme iron and the degree of lipid peroxidation were measured in liver mitochondria isolated from rats injected with either Jectofer (an iron-sorbitol-citric acid complex) or iron-nitrilotriacetate. 2. The sedimentation profiles of the mitochondria from controls and iron-treated rats as revealed by analytical differential centrifugation, indicated single population of mitochondria with $\text{s}{}_{\mathrm{4,B}}$ values of 13200± 560 S and 14200±590 S for controls and iron-loaded animals, respectively. In contrast, the sedimentation profiles of the acid phosphatase activity and the non-heme iron revealed marked polydispersities with at least three populations of particles for both controls and iron-loaded animals. 3. The mitochondria and iron-rich lysosomes were separated by density-gradient centrifugation in an isotonic medium of Percoll and sucrose. With this technique, the amount of non-heme iron in a mitochondrial fraction by differential centrifugation decreased from 69±28 nmol/mg protein to 5.6±1.1 nmol/mg protein and from 19.3±5.6 nmol/mg protein to 3.3±0.6 nmol/mg protein for Jectofer and iron-nitrilotriacetate injected rats, respectively. For control rats the amount of mitochondrial non-heme iron was about 2.7 nmol/mg protein both before and following density gradient centrifugation. The extra amount of non-heme iron still present in the purified mitochondrial fraction from iron-loaded rats, as compared to controls, was further characterized by the reactivity towards bathophenanthroline sulfonate. The results suggest that the extra iron was due to a small amount of either ferritin or hemosiderin still contaminaning the mitochondrial fraction. The amount of mitochondrial heme iron was the same in iron-loaded rats and controls. 4. The degree of lipid peroxidation in the mitochondria was estimated from the amount of malondialdehyde. The thiobarbituric acid method used for the quantitation of malondialdehyde was modified so that it was insensitive to variable amounts of iron present in the samples. No difference in the degree of lipid peroxidation was observed between the mitochondria from iron-loaded rats and controls. 5. In contrast to recent proposals (Hanstein, E.G. et al. (1981) Biochim. Biophys. Acta 678, 293–299), the present study showed that the amounts of non-heme iron and the degrees of lipid peroxidation are the same in mitochondria isolated from iron-loaded and control animals.     

Effect of Dietary Iron Loading on Recognition Memory in Growing Rats

While nutritional and neurobehavioral problems are associated with both iron deficiency during growth and overload in the elderly, the effect of iron loading in growing ages on neurobehavioral performance has not been fully explored. To characterize the role of dietary iron loading in memory function in the young, weanling rats were fed iron-loading diet (10,000 mg iron/kg diet) or iron-adequate control diet (50 mg/kg) for one month, during which a battery of behavioral tests were conducted. Iron-loaded rats displayed elevated non-heme iron levels in serum and liver, indicating a condition of systemic iron overload. In the brain, non-heme iron was elevated in the prefrontal cortex of iron-loaded rats compared with controls, whereas there was no difference in iron content in other brain regions between the two diet groups. While iron loading did not alter motor coordination or anxiety-like behavior, iron-loaded rats exhibited a better recognition memory, as represented by an increased novel object recognition index (22% increase from the reference value) than control rats (12% increase; P=0.047). Western blot analysis showed an up-regulation of dopamine receptor 1 in the prefrontal cortex from iron-loaded rats (142% increase; P=0.002). Furthermore, levels of glutamate receptors (both NMDA and AMPA) and nicotinic acetylcholine receptor (nAChR) were significantly elevated in the prefrontal cortex of iron-loaded rats (62% increase in NR1; 70% increase in Glu1A; 115% increase in nAChR). Dietary iron loading also increased the expression of NMDA receptors and nAChR in the hippocampus. These results support the idea that iron is essential for learning and memory and further reveal that iron supplementation during developmental and rapidly growing periods of life improves memory performance. Our investigation also demonstrates that both cholinergic and glutamatergic neurotransmission pathways are regulated by dietary iron and provides a molecular basis for the role of iron loading in improved memory.     

The effect of iron and ethanol on rat hepatocyte collagen synthesis

1. Carbonyl iron (2.5% w/w) in rat chow was used to induce iron loading in rat hepatocytes.2. Acute exposure of cultured hepatocytes from control and iron-loaded rats to ethanol (25–100 mM) resulted in a significant inhibition of protein synthesis.3. Inhibition of protein synthesis in hepatocytes from iron-loaded rats was primarily due to impaired amino acid uptake by these cells.4. High concentrations of ethanol stimulated the rate of protein degradation by hepatocytes from iron-loaded rats.5. Acute administration of ethanol to hepatocytes from control animals did not stimulate the absolute rates of collagen biosynthesis nor induce Type I procollagen mRNA.6. Acute administration of ethanol did not inhibit procollagen synthesis.7. Iron overload induced Type I procollagen mRNA and increased the absolute rates of collagen synthesis in hepatocytes.8. These findings may be relevant for the development of hepatic fibrosis in patients with genetic hemochromatosis who consume excess ethanol.     

The effect of iron and ethanol on rat hepatocyte collagen synthesis.

1. Carbonyl iron (2.5% w/w) in rat chow was used to induce iron loading in rat hepatocytes. 2. Acute exposure of cultured hepatocytes from control and iron-loaded rats to ethanol (25-100 mM) resulted in a significant inhibition of protein synthesis. 3. Inhibition of protein synthesis in hepatocytes from iron-loaded rats was primarily due to impaired amino acid uptake by these cells. 4. High concentrations of ethanol stimulated the rate of protein degradation by hepatocytes from iron-loaded rats. 5. Acute administration of ethanol to hepatocytes from control animals did not stimulate the absolute rates of collagen biosynthesis nor induce Type I procollagen mRNA. 6. Acute administration of ethanol did not inhibit procollagen synthesis. 7. Iron overload induced Type I procollagen mRNA and increased the absolute rates of collagen synthesis in hepatocytes. 8. These findings may be relevant for the development of hepatic fibrosis in patients with genetic hemochromatosis who consume excess ethanol.     

C3G transgenic mouse models with specific expression in platelets reveal a new role for C3G in platelet clotting through its GEF activity

We have generated mouse transgenic lineages for C3G (tgC3G) and C3GΔCat (tgC3GΔCat, C3G mutant lacking the GEF domain), where the transgenes are expressed under the control of the megakaryocyte and platelet specific PF4 (platelet factor 4) gene promoter. Transgenic platelet activity has been analyzed through in vivo and in vitro approaches, including bleeding time, aggregation assays and flow cytometry. Both transgenes are expressed (RNA and protein) in purified platelets and megakaryocytes and do not modify the number of platelets in peripheral blood. Transgenic C3G animals showed bleeding times significantly shorter than control animals, while tgC3GΔCat mice presented a remarkable bleeding diathesis as compared to their control siblings. Accordingly, platelets from tgC3G mice showed stronger activation in response to platelet agonists such as thrombin, PMA, ADP or collagen than control platelets, while those from tgC3GΔCat animals had a lower response. In addition, we present data indicating that C3G is a mediator in the PKC pathway leading to Rap1 activation. Remarkably, a significant percentage of tgC3G mice presented a higher level of neutrophils than their control siblings. These results indicate that C3G plays an important role in platelet clotting through a mechanism involving its GEF activity and suggest that it might be also involved in neutrophil development.     

Liver iron depletion and toxicity of the iron chelator Deferiprone (L, CP20) in the guinea pig

The use of the iron chelator deferiprone (L, CP20, 1,2-dimethyl-3-hydroxypyrid-4-one) for the treatment of diseases of iron overload and other disorders is problematic and requires further evaluation. In this study the efficacy, toxicity and mechanism of action of orally administered L were investigated in the guinea pig using the carbonyl iron model of iron overload. In an acute trial, depletion of liver non-heme iron in drug-treated guinea pigs (normal iron status) was maximal (approximately 50% of control) after a single oral dose of L1 of 200 mg kg, suggesting a limited chelatable pool in normal tissue. There was no apparent toxicity up to 600 mg kg. In each of two sub-acute trials, normal and iron-loaded animals were fed L (300 mg kg day) or placebo for six days. Final mortalities were 12/20 (L) and 0/20 (placebo). Symptoms included weakness, weight loss and eye discharge. Iron-loaded as well as normal guinea pigs were affected, indicating that at this drug level iron loading was not protective. In a chronic trial guinea pigs received L (50 mg kg day) or placebo for six days per week over eight months. Liver non-heme iron was reduced in animals iron-loaded prior to the trial. The increase in a wave latency (electroretinogram), the foci of hepatic, myocardial and musculo-skeletal necrosis, and the decrease in white blood cells in the drug-treated/normal diet group even at the low dose of 50 mg kg day suggests that L may be unsuitable for the treatment of diseases which do not involve Fe overload. However, the low level of pathology in animals treated with iron prior to the trial suggests that even a small degree of iron overload (two-fold after eight months) is protective at this drug level. We conclude that the relationship between drug dose and iron status is critical in avoiding toxicity and must be monitored rigorously as cellular iron is depleted.     

Iron does not cause arrhythmias in the guinea pig model of transfusional iron overload

Cardiac events, including heart failure and arrhythmias, are the leading cause of death in patients with beta thalassemia. Although cardiac arrhythmias in humans are believed to result from iron overload, excluding confounding factors in the human population is difficult. The goal of the current study was to determine whether cardiac arrhythmias occurred in the guinea pig model of secondary iron overload. Electrocardiograms were recorded by using surgically implanted telemetry devices in guinea pigs loaded intraperitoneally with iron dextran (test animals) or dextran alone (controls). Loading occurred over approximately 6 wk. Electrocardiograms were recorded for 1 wk prior to loading, throughout loading, and for approximately 4 wk after loading was complete. Cardiac and liver iron concentrations were significantly increased in the iron-loaded animals compared with controls and were in the range of those reported for humans with thalassemia. Arrhythmias were rare in both iron-loaded and control guinea pigs. No life-threatening arrhythmias were detected in either group. These data suggest that iron alone may be insufficient to cause cardiac arrhythmias in the iron-loaded guinea pig model and that arrhythmias detected in human patients with iron overload may be the result of a complex interplay of factors.     

Antioxidant and lysosomotropic properties of acute D-propranolol underlies its cardioprotection of postischemic hearts from moderate iron-overloaded rats

The benefits of acute D-propranolol (D-Pro, non-beta-adrenergic receptor blocker) pretreatment against enhanced ischemia/reperfusion (I/R) injury of hearts from moderate iron-overloaded rats were examined. Perfused hearts from iron-dextran-treated rats (450 mg/kg/week for 3 weeks, intraperitoneal administration) exhibited normal control function, despite iron treatment that elevated plasma iron and conjugated diene levels by 8.1-and 2.5-fold, respectively. However, these hearts were more susceptible to 25 mins of global I/R stress compared with non-loaded hearts; the coronary flow rate, aortic output, cardiac work, left ventricular systolic pressure, positive differential left ventricular pressure (dP/dt), and left ventricular developed pressure displayed 38%, 60%, 55%, 13%, 41%, and 15% lower recoveries, respectively, and a 6.5-fold increase in left ventricular end-diastolic pressure. Postischemic hearts from iron-loaded rats also exhibited 5.6-, 3.48-, 2.43-, and 3.45-fold increases in total effluent iron content, conjugated diene levels, lactate dehydrogenase (LDH) activity, and lysosomal N-acetyl-beta-glucosaminidase (NAGA) activity, respectively, compared with similarly stressed non-loaded hearts. A comparison of detection time profiles during reperfusion suggests that most of the oxidative injury (conjugated diene) in hearts from iron-loaded rats occurred at later times of reperfusion (8.5-15 mins), and this corresponded with heightened tissue iron and NAGA release. D-Pro (2 microM infused for 30 mins) pretreatment before ischemia protected all parameters compared with the untreated iron-loaded group; pressure indices improved 1.2- to 1.6-fold, flow parameters improved 1.70- to 2.96-fold, cardiac work improved 2.87-fold, and end-diastolic pressure was reduced 56%. D-Pro lowered total release of tissue iron, conjugated diene content, LDH activity, and NAGA activity 4.59-, 2.55-, 3.04-, and 4.14-fold, respectively, in the effluent of I/R hearts from the iron-loaded group. These findings suggest that the enhanced postischemic dysfunction and tissue injury of hearts from iron-loaded rats was caused by excessive iron-catalyzed free radical stress, and that the membrane antioxidant properties of D-Pro and its stabilization of sequestered lysosomal iron by D-Pro may contribute to the cardioprotective actions of D-Pro.     

Vitamin C suppresses oxidative lipid damage in vivo, even in the presence of iron overload

Ascorbate is a strong antioxidant; however, it can also act as a prooxidant in vitro by reducing transition metals. To investigate the in vivo relevance of this prooxidant activity, we performed a study using guinea pigs fed high or low ascorbate doses with or without prior loading with iron dextran. Iron-loaded animals gained less weight and exhibited increased plasma beta-N-acetyl-D-glucosaminidase activity, a marker of tissue lysosomal membrane damage, compared with control animals. The iron-loaded animals fed the low ascorbate dose had decreased plasma alpha-tocopherol levels and increased plasma levels of triglycerides and F(2)-isoprostanes, specific and sensitive markers of in vivo lipid peroxidation. In contrast, the two groups of animals fed the high ascorbate dose had significantly lower hepatic F(2)-isoprostane levels than the groups fed the low ascorbate dose, irrespective of iron load. These data indicate that 1) ascorbate acts as an antioxidant toward lipids in vivo, even in the presence of iron overload; 2) iron loading per se does not cause oxidative lipid damage but is associated with growth retardation and tissue damage, both of which are not affected by vitamin C; and 3) the combination of iron loading with a low ascorbate status causes additional pathophysiological changes, in particular, increased plasma triglycerides.     

Dietary supplementation with (R)-alpha-lipoic acid reverses the age-related accumulation of iron and depletion of antioxidants in the rat cerebral cortex

Accumulation of divalent metal ions (e.g. iron and copper) has been proposed to contribute to heightened oxidative stress evident in aging and neurodegenerative disorders. To understand the extent of iron accumulation and its effect on antioxidant status, we monitored iron content in the cerebral cortex of F344 rats by inductively coupled plasma atomic emission spectrometry (ICP-AES) and found that the cerebral iron levels in 24-28-month-old rats were increased by 80% (p<0.01) relative to 3-month-old rats. Iron accumulation correlated with a decline in glutathione (GSH) and the GSH/GSSG ratio, indicating that iron accumulation altered antioxidant capacity and thiol redox state in aged animals. Because (R)-alpha-Lipoic acid (LA) is a potent chelator of divalent metal ions in vitro and also regenerates other antioxidants, we monitored whether feeding LA (0.2% [w/w]; 2 weeks) could lower cortical iron and improve antioxidant status. Results show that cerebral iron levels in old LA-fed animals were lower when compared to controls and were similar to levels seen in young rats. Antioxidant status and thiol redox state also improved markedly in old LA-fed rats versus controls. These results thus show that LA supplementation may be a means to modulate the age-related accumulation of cortical iron content, thereby lowering oxidative stress associated with aging.     

Lipid peroxidation and associated hepatic organelle dysfunction in iron overload

Iron overload can have serious health consequences. Since humans lack an effective means to excrete excess iron, overload can result from an increased absorption of dietary iron or from parenteral administration of iron. When the iron burden exceeds the body's capacity for safe storage, the result is widespread damage to the liver, heart and joints, and the pancreas and other endocrine organs. Clear evidence is now available that iron overload leads to lipid peroxidation in experimental animals, if sufficiently high levels of iron are achieved. In contrast, there is a paucity of data regarding lipid peroxidation in patients with iron overload. Data from experiments using an animal model of dietary iron overload support the concept that iron overload results in an increase in an hepatic cytosolic pool of low molecular weight iron which is catalytically active in stimulating lipid peroxidation. Lipid peroxidation is associated with hepatic mitochondrial and microsomal dysfunction in experimental iron overload, and lipid peroxidation may underlie the increased lysosomal fragility that has been detected in homogenates of liver samples from both iron-loaded human subjects and experimental animals. Some current hypotheses focus on the possibility that the demonstrated functional abnormalities in organelles of the iron-loaded liver may play a pathogenic role in hepatocellular injury and eventual fibrosis. The recent demonstration that hepatic fibrosis is produced in animals with long-term dietary iron overload will allow this model to be used to further investigate the relationship between lipid peroxidation and hepatic injury in iron overload.     

Disturbances of morphological parameters in blood of rats orally exposed to aluminum chloride

The aim of this work was to assess changes of morphological parameters in the blood of rats after oral (po) administration of aluminum (Al), in relation to the time and the administered dose. The experiment was performed on female Wistar rats. The animals were administered aluminum chloride (100 mg Al/kg) daily for 21 d. Morphological assays: red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), iron serum concentration (Fe), MCH, MCHC, absolute corrected reticulocyte count (ACRC), white blood cells (WBC), and platelet count (PLT) were estimated on d 3, 7, 14, and 21, both in the control group and in intoxicated rats. After wk 1 of aluminum administration we observed a decrease of RBC, HCT, HGB and serum iron concentration in the blood of rats. The increase of the platelet count was observed earlier than changes in other parameters. Investigation has proved that the exposure of rats to aluminum administered orally results in normocytic anemia.     

Dietary supplementation with (R)-α-lipoic acid reverses the age-related accumulation of iron and depletion of antioxidants in the rat cerebral cortex

Abstract

Accumulation of divalent metal ions (e.g. iron and copper) has been proposed to contribute to heightened oxidative stress evident in aging and neurodegenerative disorders. To understand the extent of iron accumulation and its effect on antioxidant status, we monitored iron content in the cerebral cortex of F344 rats by inductively coupled plasma atomic emission spectrometry (ICP-AES) and found that the cerebral iron levels in 24–28-month-old rats were increased by 80% (p<0.01) relative to 3-month-old rats. Iron accumulation correlated with a decline in glutathione (GSH) and the GSH/GSSG ratio, indicating that iron accumulation altered antioxidant capacity and thiol redox state in aged animals. Because (R)-α-Lipoic acid (LA) is a potent chelator of divalent metal ions in vitro and also regenerates other antioxidants, we monitored whether feeding LA (0.2% [w/w]; 2 weeks) could lower cortical iron and improve antioxidant status. Results show that cerebral iron levels in old LA-fed animals were lower when compared to controls and were similar to levels seen in young rats. Antioxidant status and thiol redox state also improved markedly in old LA-fed rats versus controls. These results thus show that LA supplementation may be a means to modulate the age-related accumulation of cortical iron content, thereby lowering oxidative stress associated with aging.     

Black tea,green tea,and tea polyphenols

Previous studies have shown that tea consumption can impair trace element metabolism, particularly iron status, and increase the risk of anemia in humans and animals. More recently, however, evidence has been accumulating to show that, in animals, consumption of green tea or its polyphenols is associated with a reduction of the incidence and severity of a variety of experimentally induced cancers. In this study we have monitored the growth, trace element status, including hematological parameters of weanling rats given either (1) water, (2) 1% black tea, (3) 1% green, tea, or (4) 0.2% crude green tea extract as their sole drinking fluid while consuming diets containing either adequate or low amounts of iron. With the exception of manaanese, none of the trace elements studied (iron, copper, zinc, and manganese) or the hematological indices measured were affected by the type of beverage supplied, even though the polyphenol extract was shown to chelate metals in vitro and all the animals fed the low iron diet were shown to be anemic. There appeared to be an effect of black and green teas on manganese balance in, both the first and last weeks of the study. A lower level of brain managanese was associated with green tea consumption, and a higher level of this element in the kidneys of animals fed black tea. The results demonstrate that both black and green teas and a green tea polyphenol extract do not represent a risk to animals consuming the beverages as their sole fluid intake with respect to iron availability, although the interactions with manganese deserve further study.     

Transferrin and Iron Uptake by the Brain: Effects of Altered Iron Status

Transferrin (Tf) and iron uptake by the brain were measured in rats using 59Fe-125I-Tf and 131I-albumin (to correct for the plasma content of 59Fe and 125I-Tf in the organs). The rats were aged from 15 to 63 days and were fed (a) a low-iron diet (iron-deficient) or, as control, the same diet supplemented with iron, or (b) a chow diet with added carbonyl iron (iron overload), the chow diet alone acting as its control. Iron deficiency was associated with a significant decrease and iron overload with a significant increase in brain nonheme iron concentration relative to the controls. In each dietary treatment group, the uptake of Tf and iron by the brain decreased as the rats aged from 15 to 63 days. Both Tf and iron uptake were significantly greater in the iron-deficient rats than in their controls and lower in the iron-loaded rats than in the corresponding controls. Overall, iron deficiency produced about a doubling and iron overload a halving of the uptake values compared with the controls. In contrast to that in the brain, iron uptake by the femurs did not decrease with age and there was relatively little difference between the different dietary groups. 125I-Tf uptake by the brains of the iron-deficient rats increased very rapidly after injection of the labelled proteins, within 15 min reaching a plateau level which was maintained for at least 6 h. The uptake of 59Fe, however, increased rapidly for 1 h and then more slowly, and in terms of percentage of injected dose reached much higher values than did 125I-Tf uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of phlebotomy on the ferritin iron content in the rat liver as determined morphometrically with the use of electron energy loss spectroscopy

Summary Phlebotomy of untreated and iron-loaded rats results in a significant decrease in total liver iron. In ironloaded rats a marked decrease in iron-containing particles is observed ultrastructurally in lysosomes and cytoplasm of hepatic sinusoidal cells but not in parenchymal cells. This remarkable phenomenon was further investigated in a morphometric study, based on element-specific (iron) distribution images made in situ in the parenchymal cell by means of electron energy loss spectroscopy. With the use of this technique it could be shown that in spite of phlebotomy the ferritin iron content of the iron-loaded liver parenchymal cell is not decreased.     

Biochemical and biophysical investigations of the ferrocene-iron-loaded rat. An animal model of primary haemochromatosis.

Male Wistar rats fed with ferrocene had high hepatic iron loading (7.24 +/- 1.97 mg Fe/g tissue) after 6 weeks, principally located in lysosomes, which was comparable to the levels and distribution determined in human haemochromatosis. The two iron-storage proteins, ferritin and haemosiderin were isolated from the livers of the ferrocene-loaded rats and their iron cores were investigated by M?ssbauer spectroscopy and inductively coupled plasma-emission spectrometry. Ferrihydrite was the predominant form of iron present in both ferritin and haemosiderin, while haemosiderin contained higher amounts of phosphorus, magnesium, calcium and barium, then either normal or ferrocene-loaded ferritin. Free-radical-mediated damage in the iron-loaded livers was inferred by the significant depletion of alpha-tocopherol in both the livers and subcellular hepatic lysosomal fraction, which inversely correlated with the increasing iron content (r = -0.61; P less than 0.05) and was associated with increased fragility of the lysosomal membranes.     

Anthocyanins from red cabbage extract — evidence of protective effects on blood platelets

Red cabbage belongs to cruciferous vegetables recognized as a rich source of anthocyanins. Anthocyanins have a wide range of therapeutic advantages without adverse effects, including cardiovascular protective properties. For development of cardiovascular diseases, platelet activation is crucial; therefore compounds which inhibit platelet activation are sought after. The anti-platelet activity of anthocyanins has only been described and is still unclear. In our study, the extract of anthocyanins, obtained from fresh leaves of red cabbage, was used in vitro to examine their antioxidative effects on platelets under oxidative stress conditions which are responsible for hyperactivity of these cells. The antiplatelet and antioxidative activities were determined by platelet aggregation and specific markers of the arachidonate cascade with O₂^−· generation, and oxidative changes (carbonyl groups and 3-nitrotyrosine). Extracts (5–15 μM) protected platelet proteins and lipids against oxidative damage, and diminished platelet activation. Anthocyanins from red cabbage provided beneficial anti-platelet effects and might help prevent cardiovascular diseases.