Hepatic iron content corresponds with the susceptibility of lymphocytes to oxidative stress in neonatal pigs

The pig is born with limited iron supplies. If not supplemented, piglets dramatically loose their body iron stores during the first few days of postnatal life. The aim of this study was to investigate the influence of hepatic iron content on susceptibility of blood cells to oxidative stress. Four 1-day-old and three 7-days-old animals were used in this study. The alkaline version of the comet assay was used to measure DNA damage. As expected, iron body stores of non-supplemented animals decrease significantly during the first 4 days of life. However, no difference in background DNA damage was found between untreated lymphocytes from these two groups of animals, despite the difference in their hepatic iron content. Interestingly, DNA damage induced by H₂O₂ and X-radiation in lymphocytes taken from 1-day-old piglets was significantly higher than in those taken from 7-days-old animals. In contrast, NaOCl or tert-butyl-hydroxide also induced significant amounts of DNA damage, but no differences between the two groups of piglets were found. Our data show that decreased hepatic iron content corresponds with decreased susceptibility of blood lymphocytes to oxidative stressors.     

Assessment of oxidative stress in lymphocytes with exercise

This study investigated whether changes in the cellular composition of blood during exercise could partly account for observations of exercise-induced changes in lymphocyte oxidative stress markers. Markers of oxidative stress were assessed before and after 60 min of intense treadmill running. Samples were collected from 16 men (means ± SD: age 33 ± 13 yr; body mass index 23.8 ± 2.5 kg/m(2); maximal oxygen uptake 59.7 ± 5.2 ml·kg(-1)·min(-1)). Peripheral blood lymphocytes were assayed for protein carbonyl concentration, and plasma was assessed for lipid peroxides and antioxidant capacity. In a separate study, intracellular thiol concentration was determined in lymphocyte subsets from eight characteristically similar men by flow cytometry, of which T-cell memory populations were further identified on the basis of CD27, CD28, and CD45RA expression. Total lymphocyte protein carbonyls were transiently increased with exercise and returned to baseline within 15 min (P < 0.001). This change was accompanied by an increase in plasma lipid peroxides (P < 0.05) and total antioxidant capacity (P < 0.001). Correlation analyses showed that lymphocyte protein carbonyl content was not related to changes in the cellular composition of peripheral blood during exercise. Natural killer cells (CD3(-)CD56(+)) and late-differentiated/effector memory cells (CD4(+)/CD8(+)CD27(-)CD28(-)/CD45RA(+)), which mobilized most with exercise, showed high intracellular thiol content (P < 0.001). High thiol content suggests a lower oxidative load carried by these lymphocytes. Thus vigorous exercise resulted in a transient increase in lymphocyte oxidative stress. Results suggest this was unrelated to the alterations in the cellular composition of peripheral blood.     

Measurement of exercise-induced oxidative stress in lymphocytes

Vigorous exercise is associated with oxidative stress, a state that involves modifications to bodily molecules due to release of pro-oxidant species. Assessment of such modifications provides non-specific measures of oxidative stress in human tissues and blood, including circulating lymphocytes. Lymphocytes are a very heterogeneous group of white blood cells, consisting of subtypes that have different functions in immunity. Importantly, exercise drastically changes the lymphocyte composition in blood by increasing the numbers of some subsets, while leaving other cells unaffected. This fact may imply that observed changes in oxidative stress markers are confounded by changes in lymphocyte composition. For example, lymphocyte subsets may differ in exposure to oxidative stress because of subset differences in cell division and the acquisition of cytotoxic effector functions. The aim of the present review is to raise awareness of interpretational issues related to the assessment of oxidative stress in lymphocytes with exercise and to address the relevance of lymphocyte subset phenotyping in these contexts.     

Battles with iron: manganese in oxidative stress protection

The redox-active metal manganese plays a key role in cellular adaptation to oxidative stress. As a cofactor for manganese superoxide dismutase or through formation of non-proteinaceous manganese antioxidants, this metal can combat oxidative damage without deleterious side effects of Fenton chemistry. In either case, the antioxidant properties of manganese are vulnerable to iron. Cellular pools of iron can outcompete manganese for binding to manganese superoxide dismutase, and through Fenton chemistry, iron may counteract the benefits of non-proteinaceous manganese antioxidants. In this minireview, we highlight ways in which cells maximize the efficacy of manganese as an antioxidant in the midst of pro-oxidant iron.     

Genetic polymorphisms in the oxidative stress pathway and susceptibility to non-Hodgkin lymphoma

Oxidative damage caused by reactive oxygen species (ROS) and other free radicals is involved in a number of pathological conditions including cancer. In a population-based case-control study of non-Hodgkin lymphoma (NHL) (n = 518 cases, 597 controls) among women in Connecticut, we analyzed one or more single nucleotide polymorphisms (SNPs) in ten candidate genes (AKR1A1, AKR1C1, AKR1C3, CYBA, GPX1, MPO, NOS2A, NOS3, OGG1, and SOD2) that mediate oxidative stress directly or indirectly in the NADPH oxidase-dependent respiratory burst. Odds ratios (OR) and 95% confidence intervals (CI) were adjusted for age and race. Polymorphisms in AKR1A1 and CYBA were significantly associated with increased risk of NHL. There was a 1.7-fold (95% CI = 1.2–2.4, P = 0.0047) increased risk of NHL for individuals who were variant homozygous for the AKR1A1 (IVS5 + 282T > C) SNP. The effect was most pronounced for risk of diffuse large B-cell lymphoma, but risk estimates were non-significantly elevated for other common B-cell histologies and T-cell lymphomas as well. In addition, individuals variant homozygous for the CYBA (Ex4 + 11C > T) SNP had a 1.6-fold (95% CI = 1.1–2.4, P = 0.019) increased risk of NHL that was particularly pronounced for T-cell lymphoma (OR = 3.5, 95% CI = 1.3–9.6, P = 0.013), but was also associated with non-significant increased risks for each of the common B-cell histologies. These results suggest that SNPs in genes related to the oxidative stress pathway may be associated with increased risk of NHL. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. The US Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa

The effects of routine sperm work are often overlooked. We assessed the effect of washing cryopreserved epididymal spermatozoa from red deer (Cervus elaphus hispanicus, Helzheimer 1909). After thawing, epididymal samples (four stags) were diluted in TALP-HEPES. A split was left untouched, another was centrifuged (300 × g, 5 min) and resuspended, and a third was centrifuged and the supernatant substituted by fresh TALP-HEPES (washing). Each split was supplemented either with nothing, 1 mM of the antioxidant Trolox, 100 μM of the oxidant Fe (with ascorbate), or both. The 3 × 4 treatments were incubated at 37°C and assessed each hour up to 3 h for motility (computer-aided sperm assessment) and viability/apoptosis plus mitochondrial status (YO-PRO-1, propidium iodide, Mitotracker Deep Red; flow cytometry). DNA damage at 4 h was assessed using the terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling assay. Centrifugation alone affected neither sperm quality nor DNA, and the oxidant had no effect in control or centrifuged samples. Washed samples were not different than control, but oxidant decreased motility, mitochondrial status and viability, and altered the motility subpopulation pattern, being partially suppressed by Trolox. Spermatozoa with damaged DNA dramatically increased in the washed-oxidized sample (from 22.30 ± 3.52% to 67.94 ± 5.07%), but not when antioxidant was present. Although samples from different males behaved similarly, male-to-male variability was detected regarding susceptibility to oxidative damage after washing. We concluded that, although red deer thawed spermatozoa seemed resilient to centrifugation, the vulnerability to oxidative stress after washing makes it advisable to supplement manipulation media with antioxidants, especially taking into account male-to-male variability.     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.     

The iron cycle and oxidative stress in the lung

Iron is critical for many aspects of cellular function, but it can also generate reactive oxygen species that can damage biological macromolecules. To limit oxidative stress, iron acquisition and its distribution must be tightly regulated. In the lungs, which are continuously exposed to the atmosphere, the risk of oxidative damage is particularly high because of the high oxygen concentration and the presence of significant amounts of catalytically active iron in atmospheric particulates. An effective system of metal detoxification must exist to minimize the associated generation of oxidative stress in the lungs. Here we summarize the evidence that a number of specific proteins that control iron uptake in the gastrointestinal tract are also employed in the lung to transport iron into epithelial cells and sequester it in a catalytically inactive form in ferritin. Furthermore, these and other proteins facilitate ferritin release from lung cells to the epithelial and bronchial lining fluids for clearance by the mucociliary system or to the reticuloendothelial system for long-term storage of iron. These pathways seem to be the primary mechanism for control of oxidative stress presented by iron in the respiratory tract.     

Unincorporated iron pool is linked to oxidative stress and iron levels in Caenorhabditis elegans

Free radicals or reactive oxygen species (ROS) are relatively short-lived and are difficult to measure directly; so indirect methods have been explored for measuring these transient species. One technique that has been developed using Escherichia coli and Saccharomyces cerevisiae systems, relies on a connection between elevated superoxide levels and the build-up of a high-spin form of iron (Fe(III)) that is detectable by electron paramagnetic resonance (EPR) spectroscopy at g?=?4.3. This form of iron is referred to as "free" iron. EPR signals at g?=?4.3 are commonly encountered in biological samples owing to mononuclear high-spin (S?=?5/2) Fe(III) ions in sites of low symmetry. Unincorporated iron in this study refers to this high-spin Fe(III) that is captured by desferrioxamine which is detected by EPR at g value of 4.3. Previously, we published an adaptation of Fe(III) EPR methodology that was developed for Caenorhabditis elegans, a multi-cellular organism. In the current study, we have systematically characterized various factors that modulate this unincorporated iron pool. Our results demonstrate that the unincorporated iron as monitored by Fe(III) EPR at g?=?4.3 increased under conditions that were known to elevate steady-state ROS levels in vivo, including: paraquat treatment, hydrogen peroxide exposure, heat shock treatment, or exposure to higher growth temperature. Besides the exogenous inducers of oxidative stress, physiological aging, which is associated with elevated ROS and ROS-mediated macromolecular damage, also caused a build-up of this iron. In addition, increased iron availability increased the unincorporated iron pool as well as generalized oxidative stress. Overall, unincorporated iron increased under conditions of oxidative stress with no change in total iron levels. However, when total iron levels increased in vivo, an increase in both the pool of unincorporated iron and oxidative stress was observed suggesting that the status of the unincorporated iron pool is linked to oxidative stress and iron levels.     

Hepatic oxidative DNA damage correlates with iron overload in chronic hepatitis C patients

Hepatic oxidative stress occurs in chronic hepatitis C (CH-C), but little is known about its producing mechanisms and precise role in the pathogenesis of the disease. To determine the relevance of hepatic oxidatively generated DNA damage in CH-C, 8-hydroxy-2'-deoxyguanosine (8-OHdG) adducts were quantified in liver biopsy specimens by immunohistochemical staining, and its relationship with clinical, biochemical, and histological parameters, and treatment response was assessed in 40 CH-C patients. Hepatic 8-OHdG counts were significantly correlated with serum transaminase levels (r=0.560, p=0.0005) and histological grading activity (p=0.0013). Remarkably, 8-OHdG levels were also significantly related to body and hepatic iron storage markers (vs serum ferritin, r=0.565, p=0.0004; vs hepatic total iron score, r=0.403, p=0.0119; vs hepatic hepcidin messenger RNA, r=0.516, p=0.0013). Baseline hepatic oxidative stress was more prominent in nonsustained virological responder (non-SVR) than in SVR to interferon (IFN)/ribavirin treatment (50.8 vs 32.7 cells/10(5) microm2, p=0.0086). After phlebotomy, hepatic 8-OHdG levels were significantly reduced from 53.4 to 21.1 cells/10(5) microm2 (p=0.0125) with concomitant reductions of serum transaminase and iron-related markers in CH-C patients. In conclusion, this study showed that hepatic oxidatively generated DNA damage frequently occurs and is strongly associated with increased iron deposition and hepatic inflammation in CH-C patients, suggesting that iron overload is an important mediator of hepatic oxidative stress and disease progression in chronic HCV infection.     

Myosin heavy chain in avian muscular dystrophy corresponds to the neonatal isozyme

We have previously demonstrated, based on comparison of homologous amino acid sequences and of two-dimensional CNBr peptide gel patterns, that the myosin heavy chain in pectoralis muscles of Storrs, Connecticut dystrophic chickens is different from that of their normal controls (Huszar, G., Vigue, L., De-Lucia, J. Elzinga, M., and Haines, J. (1985) J. Biol. Chem. 260, 7429-7434). Others have shown, however, that genomic banks and mRNA complements of the control and dystrophic birds are not different. In the present studies, we have examined the hypothesis that the "dystrophic" myosin heavy chain is not a novel gene product, but is a developmental isozyme which is expressed in pectoralis muscles of adult chickens due to the dystrophic process. Two-dimensional maps of myosin heavy chain CNBr peptides were prepared from breast muscles of 17-day in ovo (embryonic), 25-day posthatch (neonatal), and adult birds of the Storrs dystrophic and of two control strains. Also, myosin and actomyosin ATPase enzymatic activities of the various preparations were determined in the pH range of 5.5 to 9.0. Analysis of the peptide maps demonstrates that the embyronic, neonatal, and control adult myosin heavy chain isozymes are distinctly different gene products with only minute variations between the respective developmental isozymes in dystrophic and control muscles. However, the pectoralis myosin heavy chain of adult dystrophic birds, which is a homogeneous isozyme population by amino acid sequences and gel patterns, corresponds to that of the neonatal-type myosin heavy chain. The ATPase properties of the embryonic, neonatal, or adult pectoralis myosins and actomyosins were not different, whether the level of specific activity or the pattern of pH activation is considered. Since the mobility of neonatal chicks (primarily neonatal-type isozymes) is not restricted, the differences in myosin heavy chain structures are part of the syndrome, but not the cause of avian muscular dystrophy.     

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.     

The influence of extracorporeal circulation on the susceptibility of erythrocytes to oxidative stress

Extracorporeal circulation (ECC), a necessary and integral part of cardiac surgery, can itself induce deleterious effects in patients. The pathogenesis of diffuse damage of several tissues is multifactorial. It is believed that circulation of blood extracorporeally through plastic tubes causes a whole body inflammatory response and a severe shear stress to blood cells. The aim of this study was to evaluate the level of oxidative stress and its deleterious effect on red blood cell (RBC) before (pre-ECC), immediately after (per-ECC) and 24 h after an ECC (24 h post-ECC). Several indicators of extracellular oxidative status were evaluated. The ascorbyl free radical (AFR) was directly measured in plasma using electron spin resonance (ESR) spectroscopy and expressed with respect to vitamin C levels in order to obtain a direct index of oxidative stress. Allophycocyanin assay was also used to investigate the plasma antioxidant status (PAS). Indirect parameters of antioxidant capacities of plasma such as vitamin E, thiol and uric acid levels were also quantified. RBC alterations were evaluated through potassium efflux and carbonyl levels after action of AAPH, a compound generating carbon centered free radicals. No changes in plasma uric acid and thiols levels were observed after ECC. However, vitamin E levels and PAS were decreased in per-ECC and 24 h post-ECC samples. Vitamin C levels were significantly lower in 24 h post-ECC and the AFR/ vitamin C ratio was increased. Differences in results had been noted when measurements took account of hemodilution. Increases of uric acid and thiols levels were observed after ECC. Vitamin E levels were not modified. However after hemodilution correction a significant decrease of vitamin C level was noted in 24 h post-ECC samples as compared to per-ECC sample. Whatever the way of measurement, vitamin C levels decreased suggesting the occurrence of ECC induced-oxidative stress. Concerning RBC, in the absence of AAPH, extracellular potassium remained unchanged between pre-, per- and 24 h post-ECC. AAPH induced a significant increase in extracellular potassium and carbonyls levels of RBC membranes, which was not modified by ECC. These results suggest the absence of alterations of RBC membrane during ECC despite the occurrence of disturbances in PAS. Such protection is of particular importance in a cell engaged in the transport of oxygen and suggests that RBC are equipped with mechanisms affording a protection against free radicals.     

Different susceptibility of prefrontal cortex and hippocampus to oxidative stress following chronic social isolation stress

Chronic oxidative stress plays an important role in depression. The aim of present study was to examine the stress-induced changes in serum corticosterone (CORT) levels, cytosolic protein carbonyl groups, malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide (NO) and total superoxide dismutase (SOD) activity in the prefrontal cortex versus hippocampus of male Wistar rats exposed to acute (2 h of immobilization or cold), chronic (21d of social isolation) stress, and their combination (chronic + acute stress). The subcellular distribution of nuclear factor-κB (NF-κB) and cytosolic cyclooxygenase 2 (COX-2) protein expressions were also examined. Depressive- and anxiety-like behaviors were assessed via the forced swim, sucrose preference, and marble burying tests in chronically isolated rats. Although both acute stressors resulted in elevated CORT, increased MDA in the prefrontal cortex and NF-κB activation accompanied by increased NO in the hippocampus were detected only following acute cold stress. Chronic isolation resulted in no change in CORT levels, but disabled appropriate response to novel acute stress and led to depressive- and anxiety-like behaviors. Increased oxidative/nitrosative stress markers, likely by NF-κB nuclear translocation and concomitant COX-2 upregulation, associated with decreased SOD activity and GSH levels, suggested the existence of oxidative stress in the prefrontal cortex. In contrast, hippocampus was less susceptible to oxidative damage showing only increase in protein carbonyl groups and depleted GSH. Taken together, the prefrontal cortex seems to be more sensitive to oxidative stress than the hippocampus following chronic isolation stress, which may be relevant for further research related to stress-induced depressive-like behavior.     

Lymphocyte DNA damage and oxidative stress in patients with iron deficiency anemia

Oxidant stress has been shown to play an important role in the pathogenesis of iron deficiency anemia. The aim of this study was to investigate the association between lymphocyte DNA damage, total antioxidant capacity and the degree of anemia in patients with iron deficiency anemia. Twenty-two female with iron deficiency anemia and 22 healthy females were enrolled in the study. Peripheral DNA damage was assessed using alkaline comet assay and plasma total antioxidant capacity was determined using an automated measurement method. Lymphocyte DNA damage of patients with iron deficiency anemia was significantly higher than controls (p<0.05), while total antioxidant capacity was significantly lower (p<0.001). While there was a positive correlation between total antioxidant capacity and hemoglobin levels (r=0.706, p<0.001), both total antioxidant capacity and hemoglobin levels were negatively correlated with DNA damage (r=-0.330, p<0.05 and r=-0.323, p<0.05, respectively). In conclusion, both oxidative stress and DNA damage are increased in IDA patients. Increased oxidative stress seems as an important factor that inducing DNA damage in those IDA patients. The relationships of oxidative stress and DNA damage with the severity of anemia suggest that both oxidative stress and DNA damage may, in part, have a role in the pathogenesis of IDA.