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.     

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.     

Hepatic injury in chronic iron overload. Role of lipid peroxidation

In both hereditary hemochromatosis and in the various forms of secondary hemochromatosis, there is a pathologic expansion of body iron stores due mainly to an increase in absorption of dietary iron. Excess deposition of iron in the parenchymal tissues of several organs (e.g. liver, heart, pancreas, joints, endocrine glands) results in cell injury and functional insufficiency. In the liver, the major pathological manifestations of chronic iron overload are fibrosis and ultimately cirrhosis. Evidence for hepatotoxicity due to iron has been provided by several clinical studies, however the specific pathophysiologic mechanisms for hepatocellular injury and hepatic fibrosis in chronic iron overload are poorly understood. The postulated mechanisms of liver injury in chronic iron overload include (a) increased lysosomal membrane fragility, perhaps mediated by iron-induced lipid peroxidation, (b) peroxidative damage to mitochondria and microsomes resulting in organelle dysfunction, (c) a direct effect of iron on collagen biosynthesis and (d) a combination of all of the above.     

Intrahepatic hepatitis C virus replication correlates with chronic hepatitis C disease severity in vivo

The role of viral factors in the pathogenesis of chronic hepatitis C is unknown. The objective of the present study was to characterize markers of hepatitis C virus (HCV) infection and replication in liver biopsy specimens obtained from 65 genotype 1-infected subjects, including 31 who were coinfected with human immunodeficiency virus (HIV), and to analyze associations between intrahepatic viral markers and hepatitis C disease severity. The percentages of liver cells harboring HCV genomes (%G) and replicative-intermediate RNAs (%RI) were evaluated using strand-specific in situ hybridization, while HCV core and NS3 antigens were assessed by immunocytochemistry. HIV-positive and HIV-negative subjects had similar mean grades and stages of liver disease and had similar indices of HCV infection and replication in liver, even though coinfected subjects had significantly shorter mean disease duration (P = 0.0003). Multivariate analysis showed that %G was not associated with grade or stage of liver disease (P = 0.5 and 0.4, respectively), while %RI was strongly associated with liver inflammation (P < 0.001), liver fibrosis (P < 0.001), and serum alanine aminotransferase levels (P = 0.01). NS3 antigen (but not core) was more frequently detected in HCV RI-positive versus RI-negative specimens (P = 0.028). These findings demonstrate a link between HCV proliferation and hepatitis C disease severity and suggest similar pathogenic mechanisms in HIV-positive and HIV-negative individuals.     

Insulin resistance is associated with DNA damage in peripheral blood cells in non-diabetic patients with genotype 1 chronic hepatitis C

Abstract

Background. In chronic liver diseases of different etiologies, including viral hepatitis, genotoxic effects of oxidative stress have been shown, both in clinical and in experimental conditions, suggesting that this mechanism may contribute to the evolution of the disease. Aim. To evaluate DNA damage in the peripheral blood of untreated non-diabetic patients with chronic hepatitis C and control subjects, and its correlation with demographic, anthropometric, biochemical, and histological parameters in the patient sample. Patients and methods. This study comprised 100 subjects of both genders, 60 of whom were treatment-naïve patients with positive serology for genotype 1 hepatitis C. The remaining 40 were blood donors with negative serology for hepatitis who were used as control subjects, and matched by gender, age, weight, and BMI. DNA damage was determined using the comet assay in the total peripheral blood. Results. The DNA damage evaluated by the comet assay revealed higher values in the group of patients with hepatitis compared with that in the control group. The relationships of the comet assay with the studied variables were assessed using multivariate analysis; significant correlations were only identified with insulin (r = 0.343, p = 0.008) and Homeostasis Model Assessment Insulin Resistance (HOMA-IR) (r = 0.331, p = 0.011). Conclusion. Patients with genotype 1 chronic hepatitis C have higher rates of DNA damage, as determined by comet assay and this alteration is correlated with the HOMA index of insulin resistance.     

Mitochondrial DNA mutations and oxidative damage in skeletal muscle of patients with chronic uremia

Abundant evidence has been gathered to suggest that mitochondrial DNA (mtDNA) sustains many more mutations and greater oxidative damage than does nuclear DNA in human tissues. Uremic patients are subject to a state of enhanced oxidative stress due to excess production of oxidants and a defective antioxidant defense system. This study was conducted to investigate mtDNA mutations and oxidative damage in skeletal muscle of patients with chronic uremia. Results showed that large-scale deletions between nucleotide position (np) 7,900 and 16,300 of mtDNA occurred at a high frequency in muscle of uremic patients. Among them, the 4,977-bp deletion (mtDNA⁴⁹⁷⁷) was the most frequent and most abundant large-scale mtDNA deletion in uremic skeletal muscle. The proportion of mtDNA⁴⁹⁷⁷ was found to correlate positively with the level of 8-hydroxy 2-deoxyguanosine (8-OHdG) in the total DNA of skeletal muscle (r=0.62, p<0.05). Using long-range PCR and DNA sequencing, we identified and characterized multiple deletions of mtDNA in skeletal muscle of 16 of 19 uremic patients examined. The 8,041-bp deletion, which occurred between np 8035 and 16,075, was flanked by a 5-bp direct repeat of 5-CCCAT-3. Some of the deletions were found in more than 1 patient. On the other hand, we found that the mean 8-OHdG/10⁵ dG ratio in the total cellular DNA of muscle of uremic patients was significantly higher than that of the controls (182.7 ± 63.6 vs. 50.9 ± 21.5, p=0.05). In addition, the mean 8-OHdG/10⁵ dG ratio in muscle mtDNA of uremic patients was significantly higher than that in nuclear DNA (344.0 ± 56.9 vs. 146.3 ± 95.8, p=0.001). Moreover, we found that the average content of lipid peroxides in mitochondrial membranes of skeletal muscle of uremic patients was significantly higher than that of age-matched healthy subjects (23.76 ± 6.06 vs. 7.67 ± 0.95 nmol/mg protein; p<0.05). The average content of protein carbonyls in the mitochondrial membranes prepared from uremic skeletal muscles was significantly higher than that in normal controls (24.90 ± 4.00 vs. 14.48 ± 1.13 nmol/mg protein; p<0.05). Taken together, these findings suggest that chronic uremia leads to mtDNA mutations together with enhanced oxidative damage to DNA, lipids, and proteins of mitochondria in skeletal muscle, which may contribute to the impairment of mitochondrial bioenergetic function and to skeletal myopathy commonly seen in uremic patients.     

Labile iron pool correlates with iron content in the nucleus and the formation of oxidative DNA damage in mouse lymphoma L5178Y cell lines

Labile iron pool (LIP) constitutes a crossroad of metabolic pathways of iron-containing compounds and is midway between the cellular need for iron, its uptake and storage. In this study we investigated oxidative DNA damage in relation to the labile iron pool in a pair of mouse lymphoma L5178Y (LY) sublines (LY-R and LY-S) differing in sensitivity to hydrogen peroxide. The LY-R cells, which are hydrogen peroxide-sensitive, contain 3 times more labile iron than the hydrogen peroxide-resistant LY-S cells. Using the comet assay, we compared total DNA breakage in the studied cell lines treated with hydrogen peroxide (25 microM for 30 min at 4 degrees C). More DNA damage was found in LY-R cells than in LY-S cells. We also compared the levels of DNA lesions sensitive to specific DNA repair enzymes in both cell lines treated with H(2)O(2). The levels of endonuclease III-sensitive sites and Fapy-DNA glycosylase-sensitive sites were found to be higher in LY-R cells than in LY-S cells. Our data suggest that the sensitivity of LY-R cells to H(2)O(2) is partially caused by the higher yield of oxidative DNA damage, as compared to that in LY-S cells. The critical factor appears to be the availability of transition metal ions that take part in the OH radical-generating Fenton reaction (very likely in the form of LIP).     

Dose-dependent increase of oxidative damage in the testes of rats subjected to acute iron overload

This study describes the in vivo response of rat testes to acute iron overload. Male Wistar rats (250-300 g) were injected ip with iron dextran at doses of 250 (Fe250), 500 (Fe500), or 1000 mg/kg body wt (Fe1000) or with saline (C). Parameters of oxidative stress and iron toxicity were measured 20 h after injection. Total iron content was 3.5-, 5.3-, and 10.4-fold higher in the Fe250, Fe500, and Fe1000 groups, respectively, compared to controls (320 +/- 22 nmol/g tissue). Histological studies showed that: (a) iron accumulated in the sperm and other testes cells, and (b) spermatogenesis was markedly lower in the Fe1000 group. The concentration of alpha-tocopherol, ubiquinol-9, and ubiquinol-10 in the testes was inversely correlated with the extent of oxidation. Testes chemiluminescence was 45% higher in the Fe1000 group compared to controls (41 cps/cm(2)). Endogenous levels of lipid oxidation, evaluated as 2-thiobarbituric acid-reactive substances, were 46, 73, and 82% higher in the groups Fe250, Fe500, and Fe1000, respectively, than in controls (33.6 +/- 1.4 nmol/g tissue). Oxidative damage to DNA evaluated by the presence of 8-oxo-2'-deoxyguanosine (oxo(8)dG), was 26, 39, and 74% higher in the Fe250, Fe500, and Fe1000 groups, respectively, than in the C group (2.3 +/- 0.1 oxo(8)dG/10(5)dG). Protein oxidation was measured as protein thiols and carbonyl content in proteins and glutamine synthase activity. Protein thiols content and glutamine synthase activity were similar in all the groups, while the protein-associated carbonyls content was 96% higher in the Fe1000 group than in the C group (2.1 +/- 0.4 nmol/mg protein). No changes in the activities of superoxide dismutase, catalase, and glutathione peroxidase were observed. The results showed that in vivo iron overload induced oxidative stress and the impairment of spermatogenesis in rat testes that were dependent on the amount of iron supplemented and its accumulation in the tissue.     

Increased oxidative damage to DNA in ALS patients

Although the cause of amyotrophic lateral sclerosis (ALS) is unknown, substantial evidence indicates that oxidative toxicity is associated with neuronal death in this disease. We examined levels of a well-established marker of oxidative damage to DNA, 8-hydroxy-2'-deoxyguanosine (8OH2'dG) in plasma, urine, and cerebrospinal fluid (CSF) at a single time point from subjects with ALS, other neurological diseases, or no known disorders. We also measured the rate of change of 8OH2'dG levels in plasma and urine from ALS and in urine from control subjects over 9 months and examined the relationship to disease severity. In each fluid, 8OH2'dG levels were significantly elevated in the ALS group as compared to control subjects. In all subjects, the plasma and CSF 8OH2'dG levels increased with age, providing further evidence for a role of oxidative damage in normal aging. Plasma and urine 8OH2'dG levels increased significantly with time in the ALS group only. The rate of increase in urine 8OH2'dG levels with time was significantly correlated with disease severity. These findings are consistent with the hypothesis that oxidative pathology accompanies the neurodegenerative process in ALS and suggest that 8OH2'dG may provide a useful tool for monitoring therapeutic interventions in this disease.     

Body iron is a contributor to oxidative damage of DNA

The transition metal iron is catalytically highly active in vitro, and not surprisingly, body iron has been suggested to promote oxidative stress in vivo. In the current analysis we studied the association of serum ferritin concentration and serum soluble transferrin receptor concentration with daily urinary 8-hydroxydeoxyguanosine excretion, a marker of oxidative stress, in 48 mildly dyslipidemic men in East Finland. In multivariate linear regression analyses allowing for age, smoking, body mass index and physical exercise, serum ferritin concentration predicted the excretion rate at B = 0.17 (95% CI 0.08–0.26, P = 0.001), and serum soluble transferrin receptor to ferritin concentration ratio (TfR/ferritin) predicted the excretion rate at B = ? 0.13 (95% CI ? 0.21 to ? 0.05, P = 0.002). Our data suggest that body iron contributes to excess oxidative stress already at non-iron overload concentrations in these subjects.     

Body iron is a contributor to oxidative damage of DNA

The transition metal iron is catalytically highly active in vitro, and not surprisingly, body iron has been suggested to promote oxidative stress in vivo. In the current analysis we studied the association of serum ferritin concentration and serum soluble transferrin receptor concentration with daily urinary 8-hydroxydeoxyguanosine excretion, a marker of oxidative stress, in 48 mildly dyslipidemic men in East Finland. In multivariate linear regression analyses allowing for age, smoking, body mass index and physical exercise, serum ferritin concentration predicted the excretion rate at B = 0.17 (95% CI 0.08-0.26, P = 0.001), and serum soluble transferrin receptor to ferritin concentration ratio (TfR/ferritin) predicted the excretion rate at B = - 0.13 (95% CI - 0.21 to - 0.05, P = 0.002). Our data suggest that body iron contributes to excess oxidative stress already at non-iron overload concentrations in these subjects.     

Soluble serum CD81 is elevated in patients with chronic hepatitis C and correlates with alanine aminotransferase serum activity

Aim

Cellular CD81 is a well characterized hepatitis C virus (HCV) entry factor, while the relevance of soluble exosomal CD81 in HCV pathogenesis is poorly defined. We performed a case-control study to investigate whether soluble CD81 in the exosomal serum fraction is associated with HCV replication and inflammatory activity.

Patients and Methods

Four cohorts were investigated, patients with chronic hepatitis C (n = 37), patients with chronic HCV infection and persistently normal ALT levels (n = 24), patients with long term sustained virologic response (SVR, n = 7), and healthy volunteers (n = 23). Concentration of soluble CD81 was assessed semi-quantitatively after differential centrifugation ranging from 200 g to 100,000 g in the fifth centrifugation fraction by immunoblotting and densitometry.

Results

Soluble CD81 was increased in patients with chronic hepatitis C compared to healthy subjects (p = 0.03) and cured patients (p = 0.017). Patients with chronic HCV infection and persistently normal ALT levels and patients with long term SVR had similar soluble CD81 levels as healthy controls (p>0.2). Overall, soluble CD81 levels were associated with ALT levels (r = 0.334, p = 0.016) and severe liver fibrosis (p = 0.027).

Conclusion

CD81 is increased in the exosomal serum fraction in patients with chronic hepatitis C and appears to be associated with inflammatory activity and severity of fibrosis.     

Heterogeneity in premature senescence by oxidative stress correlates with differential DNA damage during the cell cycle

The development of cellular senescence both by replication and by oxidative stress is not homogenous in cultured primary human fibroblasts. To investigate whether this is due to the heterogeneity in the susceptibility of DNA in different phases of the cell cycle, we subjected synchronised cells to oxidative stress and examined the extent of DNA damage and its long-term effects on the induction of cellular senescence. Here, we first show marked heterogeneity in DNA damage as detected by markers of double strand breaks caused by oxidative stress in an asynchronous human fibroblast culture. Cell cycle synchronization followed by oxidative stress demonstrated that DNA in S-phase is most susceptible to oxidative stress whereas DNA in the quiescent phase is most resistant. DNA repair is an ongoing process after sensing DNA damage; reparable DNA damage is repaired even in cells that contain persistent DNA damage. The extent of persistent DNA damage is tightly correlated with permanent cessation of DNA replication and SA-beta-gal activity. Oxidative stress encountered by cells in S-phase resulted in more persistent DNA damage, more permanent cell cycle arrest and the induction of premature senescence.     

Occult hepatitis C virus elicits mitochondrial oxidative stress in lymphocytes and triggers PI3-kinase-mediated DNA damage response

Occult hepatitis C viral infection (OHCI) is a newly reported pathological entity associated with increased risk of developing hepatocellular carcinoma and lymphoproliferative disorders. Although hepatocytes are the primary sites of viral replication, hepatitis C virus is potentially lymphotropic, invading and propagating in cells of the immune system. Lymphocytes, the extrahepatic viral reservoirs, are differentially implicated in the occult and the active forms of the disease. This study aimed to elucidate the implications of mitochondrial oxidative stress on the immune pathophysiological mechanisms of OHCI. We herein report that OHCI induces mitochondrial oxidative stress, leading to DNA double-strand breaks and elicitation of a phosphoinositol 3-kinase-mediated cellular response in peripheral blood lymphocytes. Compared to controls, OHCI subjects showed higher accumulation of pATM, pATR, γH2AX, and p-p53, along with active recruitment of repair proteins (Mre11, Rad50, and Nbs1) and altered mitochondrial DNA content. Increased mitochondrial membrane depolarization and circulating nucleosome levels along with chromatid-type aberrations and decreased T-cell proliferative index observed in the OHCI group further indicated that this damage might lead to Bax-triggered mitochondria-mediated cellular apoptosis. Together our results provide the mechanistic underpinnings of mitochondrial dysfunction in OHCI, a previously unknown paradigm, for explaining the immune pathogenesis in a redox-dependent manner.     

Co-infections with hepatitis G and TT virus in patients with chronic hepatitis C in Hungary

The significance of co-infections with novel hepatitis viruses Hepatitis G (GBV-C, HGV) and TT virus (TTV) in chronic hepatitis C is not clear. We determined the prevalence of HGV RNA and TTV DNA in chronic hepatitis C patients and in asymptomatic hepatitis C virus (HCV) carriers, and assessed the influence of these agents on the course of HCV infection. Seventy-seven patients with chronic hepatitis C--50 of them treated with interferon (IFN)--and 33 HCV carriers with normal alanine aminotransferase have been investigated. Previous HBV infection was detected by testing serum HBsAg and aHBc. HGV RNA and TTV DNA were detected by PCR. In the healthy population, the prevalence of anti-HCV was 0.3%, HGV RNA 8.0% and TTV DNA 18.5%. In chronic hepatitis C HGV RNA occurred in 9.09% and TTV DNA in 40.25% of cases. In IFN-treated patients with sustained remission, the frequency of TTV was 20% vs. 45.7% found in non-responders. Among asymptomatic HCV-carriers, the prevalence of HGV RNA was 9.09% and TTV DNA 75.7%. Neither HGV RNA nor TTV DNA had apparent effect on the HCV infection. TTV was detected with the lowest frequency in persons with sustained remission due to IFN, suggesting antiviral effect of IFN on TTV.     

Levels of some cytokines and antibodies to hepatitis C virus in patients with chronic hepatitis C

The comparison of the levels of some cytokines (tumor necrosis factor alpha (TNF-alpha), IL-1beta, IL-2, IL-4) in the blood serum of patients with chronic hepatitis C (CHC) having different antibody spectrum was carried out. In CHC patients increased levels of the serum cytokines IL-1beta, TNF-alpha under study in comparison with cytokine levels in donor sera was noted. In patients with detected antiNS5 and antiHCV IgM and antiNS5 HCV the level of IL-1beta was significantly higher than that in CHC patients without antibodies in sera. A change in the levels of proinflammatory and anti-inflammatory cytokines in the blood sera of CHC patients may be of significant diagnostic and prognostic importance.     

Elevated hepatic iron: A confounding factor in chronic hepatitis C

Historically, iron overload in the liver has been associated with the genetic disorders hereditary hemochromatosis and thalassemia and with unusual dietary habits. More recently, elevated hepatic iron levels also have been observed in chronic hepatitis C virus (HCV) infection. Iron overload in the liver causes many changes including induction of oxidative stress, damage to lysosomes and mitochondria, altered oxidant defense systems and stimulation of hepatocyte proliferation. Chronic HCV infection causes numerous pathogenic changes in the liver including induction of endoplasmic reticulum stress, the unfolded protein response, oxidative stress, mitochondrial dysfunction and altered growth control. Understanding the molecular and cellular changes that could occur in a liver which has elevated hepatic iron levels and in which HCV replication and gene expression are ongoing has clinical relevance and represents an area of research in need of further investigation.