Systemic oxidative stress in children and teenagers with Down syndrome

Aims

The aim of this study was to evaluate the antioxidant status and oxidative stress biomarkers in the blood of children and teenagers with Down syndrome.

Main methods

The analysis of enzymatic antioxidant defenses, such as the activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione transferase (GST), non-enzymatic antioxidants, such as levels of reduced glutathione (GSH), uric acid (UA) and vitamin E, as well as oxidative damage indicators, such as protein carbonyls (PC) levels and lipoperoxidation (TBARS), of DS individuals (n = 20) compared to healthy controls (n = 18). Except the vitamin E was measured by HPLC, all other markers were measured spectrophotometrically.

Key Findings

Antioxidant enzymes analysis showed significant increases in the SOD (47.2%), CAT (24.7%) and GR (49.6%) activities in DS subjects. No significant difference in GPx activity was detected while GST activity (61.2%) was decreased, and both responses may be consequence of the depletion of GSH (24.9%) levels. There were no significant differences in TBARS levels, while PC levels showed decreased (31.7%) levels compared to healthy controls, which may be related to the increase (16.1%) found in serum UA. Levels of vitamin E showed no significant differences between DS individuals compared to controls.

Significance

The results revealed a systemic pro-oxidant status in DS individuals, evidenced by the increased activity of some important antioxidant enzymes, together with decreased GSH levels in whole blood and elevated UA levels in plasma, probably as an antioxidant compensation related to the redox imbalance in DS individuals.     

Fast quantification of the urinary marker of oxidative stress 8-hydroxy-2′-deoxyguanosine using solid-phase extraction and high-performance liquid chromatography with triple-stage quadrupole mass detection

Exogenous and endogenous oxidants constantly cause oxidative damage to DNA. Since the reactive oxidants itself are not suitable for analysis, oxidized bases like 8-hydroxy-2′-deoxyguanosine (8OHdG) are used as biomarkers for oxidative stress, either in cellular DNA or as elimination product in urine. A simple, fast and robust analytical procedure is described for urinary 8OHdG as an indicator of oxidative damage in humans. The adduct was purified from human urine by applying a single solid-phase extraction step on LiChrolut EN^®. After evaporation of the eluate, the residue was resolved and an aliquote was injected into a HPLC system with a triple quadrupole mass spectrometer. The limit of detection was 0.2 ng ml⁻¹ (7 fmol absolute) when using one product ion as quantifier and two further product ions as qualifier. The coefficient of variation was 10.1% (n=5 at 2.8 ng ml⁻¹ urine). The sample throughput was about 50 samples a day. Thus, this method is more sensitive and much faster than the common method using HPLC with electrochemical detection. The results of a study with nine volunteers investigated at six time-points each over 5 days are presented. The mean excretion of 8OHdG was 2.1 ng mg⁻¹ creatinine (range 0.17–5.9 ng mg⁻¹ creatinine; 4 of 53 samples were below the LOD). A relatively large intra- (relative SD 66%) and inter-individual (relative SD 71%) variation in urinary 8OHdG excretion rates was found.     

Oxidative DNA damage in tissues of English sole (Parophrys vetulus) exposed to nitrofurantoin

Juvenile English sole were exposed intramuscularly to nitrofurantoin (NF) and the levels of 8-hydroxy-2′deoxyguanosine (8-OH-dG) in liver, kidney and blood were determined using reversed-phase HPLC with electrochemical detection. Identification and quantitation of the 8-OH-dG in the samples was accomplished by comparison with standard 8-OH-dG, which was characterized by UV spectroscopy and fast-atom bombardment mass spectrometry. The levels of hepatic 8-OH-dG increased (r² = 0.59, P = 0.015) with the dose of NF (0.10 – 10 mg NF/kg fish). In kidney and blood, however, the levels of 8-OH-dG were significantly higher than controls only at the highest dose tested. The level of binding in liver ranged from 0.37 to 0.76 fmol 8-OH-dG/μg DNA. The levels of hepatic 8-OH-dG reached a maximum (approx. 1 fmol 8-OH-dG/μg DNA) between 1 and 3 days after exposure, followed by a decrease to control levels (approx. 0.25 fmol 8-OH-dG/μg DNA) at 5 days post-exposure. These data demonstrate the first direct evidence for the formation of oxidized DNA bases resulting from the metabolism of a nitroaromatic compound by fish.     

Markers of macromolecular oxidative damage in maternal serum and risk of neural tube defects in offspring

Neural tube defects (NTDs) are among the most common and severe congenital malformations. To examine the association between markers of macromolecular oxidative damage and risk of NTDs, we measured levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), protein carbonyl (PC), and 8-iso-prostaglandin F2α (8-iso-PGF2α) in maternal serum samples of 117 women with NTD-affected pregnancies and 121 women with healthy term newborns. We found higher levels of 8-OHdG and PC in the NTD group than in the control group; however, we did not observe a statistically significant difference in 8-iso-PGF2α levels between the NTD and the control groups. NTD risk increased with increasing quartiles of 8-OHdG [odds ratio (OR)=1.17; 95% confidence interval (CI) 0.39–3.51; OR=2.19; 95% CI, 0.68–7.01; OR=3.70; 95% CI, 1.30–10.51, for the second, third, and fourth quartile relative to the lowest quartile, respectively; P=0.009], and with increasing quartiles of PC (OR=2.26; 95% CI, 0.66–7.69; OR=3.86; 95% CI, 1.17–12.80; OR=5.98; 95% CI, 1.82–19.66, for the second, third, and fourth quartile relative to the lowest quartile, respectively; P=0.002]. Serum levels of 8-OHdG were higher in women who did not take folic acid supplements during the periconceptional period. These results suggest that oxidative stress is present in women carrying pregnancies affected by NTDs.     

Effects of environmental air pollution on endogenous oxidative DNA damage in humans

Epidemiological studies conducted in metropolitan areas have demonstrated that exposure to environmental air pollution is associated with increases in mortality. Carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) are the major source of genotoxic activities of organic mixtures associated with respirable particulate matter, which is a constituent of environmental air pollution. In this study,we wanted to evaluate the relationship between exposure to these genotoxic compounds present in the air and endogenous oxidative DNA damage in three different human populations exposed to varying levels of environmental air pollution. As measures of oxidative DNA damage we have determined 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and cyclic pyrimidopurinone N-1,N² malondialdehyde-2′-deoxyguanosine (M₁dG) by the immunoslot blot assay from lymphocyte DNA of participating individuals. The level of endogenous oxidative DNA damage was significantly increased in individuals exposed to environmental air pollution compared to unexposed individuals from Kosice (8-oxodG adducts) and Sofia (M₁dG adducts). However, there was no significant difference in the level of endogenous oxidative DNA and exposure to environmental air pollution in individuals from Prague (8-oxodG and M₁dG adducts) and Kosice (M₁dG adducts). The average level of M₁dG adducts was significantly lower in unexposed and exposed individuals from Kosice compared to those from Prague and Sofia. The average level of 8-oxodG adducts was significantly higher in unexposed and exposed individuals from Kosice compared to those from Prague. A significant increasing trend according to the interaction of c-PAHs exposure and smoking status was observed in levels of 8-oxodG adducts in individuals from Kosice. However, no other relationship was observed for M₁dG and 8-oxodG adduct levels with regard to the smoking status and c-PAH exposure status of the individuals. The conclusion that can be made from this study is that environmental air pollution may alter the endogenous oxidative DNA damage levels in humans but the effect appears to be related to the country where the individuals reside. Genetic polymorphisms of the genes involved in metabolism and detoxification and also differences in the DNA repair capacity and antioxidant status of the individuals could be possible explanations for the variation observed in the level of endogenous oxidative DNA damage for the different populations.     

Accumulation of oxidative stress around the stroke-like lesions of MELAS patients

To investigate the relationship between oxidative stress and progressive spread of the stroke-like lesions in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) with 3243A>G mutation, we retrospectively analyzed the spread frequency in patients with and without treatment with the radical scavenger edaravone. Oxidative damage and defensive enzymes were histologically evaluated. Spread was significantly less frequent in the patients treated with edaravone. Although 8-hydroxy-2′-deoxyguanosine, a marker for oxidative damage of DNA, was obviously accumulated in peri-lesional surviving neurons, manganese superoxide dismutase and 8-oxoguanine glycosylase 1 were not up-regulated in those neurons. Increased oxidative stress and insufficient defense could be involved in the pathogenesis of the spreading lesions in MELAS.     

Oxidative stress in neonates: evaluation using specific biomarkers

Increased oxidative stress has been implicated in pathogenesis of serious diseases in neonates. We measured urinary levels of 8-hydroxy-2'-deoxyguanosine (a marker of oxidative DNA damage), acrolein-lysine adduct (a marker of lipid peroxidation and oxidative protein damage), and nitrite/nitrate (a marker of endogenous nitric oxide formation) in one-month-old neonates to examine the status of oxidative stress and its relationship to the degree of prematurity and clinical condition in neonates. Study subjects comprised three groups: healthy term neonates, clinically stable preterm neonates requiring no supplemental oxygen, and clinically sick preterm neonates requiring supplemental oxygen and ventilator support. Urinary levels of 8-hydroxy-2'-deoxyguanosine and acrolein-lysine adduct were significantly higher in sick preterm neonates than those of stable preterm and healthy term neonates. In the sick preterm group, neonates developing active retinopathy showed significantly higher levels of acrolein-lysine adduct than the other neonates without retinopathy. There were no significant differences in both urinary markers of oxidative stress between stable preterm and healthy term neonates. The urinary nitrite/nitrate levels were not significantly different among the three groups, suggesting no difference in endogenous nitric oxide formation. Collectively, these results provide evidence of augmentation of oxidative damage to DNA, lipids and proteins, especially in clinically sick preterm neonates.     

Neopterin, a marker of immune response, and 8-hydroxy-2'-deoxyguanosine, a marker of oxidative stress, correlate at high age as determined by automated simultaneous high-performance liquid chromatography analysis of human urine

Using an established high-performance liquid chromatography (HPLC) method based on anion exchange chromatography, fraction collection, and electrochemical detection, the oxidative DNA damage marker 8-hydroxy-2′-deoxyguanosine (8-OH-dG) can be analyzed rapidly and precisely in human urine samples. In addition, by ultraviolet (UV) detection, it was shown recently that it is possible to simultaneously analyze creatinine and 7-methylguanine (m⁷Gua), an RNA degradation product, in urine. By adding a fluorescence detector to the HPLC system, we now report that it is also possible to detect pteridins such as neopterin and biopterin. The fluorescence detection was evaluated in detail for neopterin, an immune response and tumor marker. The urinary content of neopterin, assessed by using the HPLC method, was verified with a commercial neopterin enzyme-linked immunosorbent assay (ELISA) kit as indicated by the high correlation between the two methods (r = 0.98). In urinary samples from 58 young healthy individuals (male and female nonsmokers, ages 19-39 years), it was found that there was no significant correlation (r = −0.04) between the levels of 8-OH-dG and neopterin (as normalized to urinary creatinine levels). In contrast, in urinary samples from 60 old healthy individuals (male and female nonsmokers, ages 60-86 years), there was a significant correlation (r = 0.47) found between the levels of 8-OH-dG and neopterin (as normalized to urinary creatinine levels). These findings strongly indicate that the higher level of immune response that was correlating with old age contributes significantly to the higher level of oxidative damage as assessed in the form of 8-OH-dG. Using this type of HPLC system, it is possible to evaluate oxidative DNA damage and immune response simultaneously using the respective urinary markers. These data may contribute to understanding of the pathophysiology of diseases such as infections and tumor progression where both oxidative stress and immune response occur simultaneously.     

Recent developments in analytical methodology for 8-hydroxy-2'-deoxyguanosine and related compounds

When biomolecules such as proteins, lipids, and DNA are subjected to oxidative attack by free radicals or other reactive species, a number of measurable biomarkers may be produced. The study of oxidative DNA damage is valuable in research concerning cancer and aging. The current review includes methodology involving various separation science techniques for the analysis of DNA oxidation biomarkers, mainly 8-hydroxy-2'-deoxyguanosine. This review will present recent analytical developments with respect to sample preparation and instrumental considerations, noting key outcomes and biological relevance where appropriate.     

Oxidative DNA damage in tissues of English sole (parophrys vetulus) exposed to nitrofurantoin

Juvenile English sole were exposed intramuscularly to nitrofurantoin (NF) and the levels of 8-hydroxy-2′deoxyguanosine (8-OH-dG) in liver, kidney and blood were determined using reversed-phase HPLC with electrochemical detection. Identification and quantitation of the 8-OH-dG in the samples was accomplished by comparison with standard 8-OH-dG, which was characterized by UV spectroscopy and fast-atom bombardment mass spectrometry. The levels of hepatic 8-OH-dG increased (r² = 0.59, P = 0.015) with the dose of NF (0.10 – 10 mg NF/kg fish). In kidney and blood, however, the levels of 8-OH-dG were significantly higher than controls only at the highest dose tested. The level of binding in liver ranged from 0.37 to 0.76 fmol 8-OH-dG/μg DNA. The levels of hepatic 8-OH-dG reached a maximum (approx. 1 fmol 8-OH-dG/μg DNA) between 1 and 3 days after exposure, followed by a decrease to control levels (approx. 0.25 fmol 8-OH-dG/μg DNA) at 5 days post-exposure. These data demonstrate the first direct evidence for the formation of oxidized DNA bases resulting from the metabolism of a nitroaromatic compound by fish.     

Trace elements,oxidative stress and glycemic control in young people with type 1 diabetes mellitus

Trace elements and oxidative stress are associated with glycemic control and diabetic complications in type 1 diabetes mellitus. In this study, we analyzed the levels of serum copper, zinc, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) and urinary MDA and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in 33 type 1 diabetic patients with optimal and suboptimal glycemic control (HbA_1C < 9.0%) and 40 patients with poor glycemic control (HbA_1C ≥ 9%) and 27 age- and sex-matched non-diabetic controls to evaluate the differences between these markers in different glycemic control states. Diabetic patients, especially poor-glycemic-control subjects (HbA_1C ≥ 9%), exhibited significantly lower levels of serum zinc and increased levels of serum copper (and, therefore, increased serum copper-to-zinc ratios), serum SOD, blood MDA, and urinary MDA and 8-OHdG, relative to non-diabetic subjects. Furthermore, significant correlations existed in these patients between the serum copper, serum copper-to-zinc ratio, and urinary MDA (all p < 0.001) and the levels of urinary 8-OHdG (p = 0.007) and HbA_1C. Our results suggest that high serum copper levels and oxidative stress correlate with glycemic control. Therefore, strict glycemic control, decreased oxidative stress, and a lower copper concentration might prevent diabetic complications in patients with type 1 diabetes mellitus.     

Biomarkers of age effect on renal function in Down syndrome

Objective: To assess differences in kidney function between Down syndrome (DS) individuals and a control group related to aging.

Methods: Creatinine (Cr) and specific gravity (SG) were assessed by spectrophotometric and refractometric assays in urine samples of 103 individuals with DS and 82 age-matched controls.

Results: Significantly lower levels of Cr and SG were found in DS after puberty. Significant correlations were found between SG and age as well as between Cr and SG in DS and controls (p?≤?0.05).

Conclusions: Premature aging in kidneys of DS patients could lead to an impaired renal function.     

Differences in oxidative stress markers based on the aetiology of heart failure: Comparison of oxidative stress in patients with and without coronary artery disease

Abstract

Various oxidative stress markers have been measured to evaluate the status of heart failure (HF). However, the relationships between these markers and the aetiology of HF have not been fully investigated. This study compared 8-hydroxy-2′-deoxyguanosine (8-OHdG) and biopyrrins levels in patients with ischemic and non-ischemic HF. Study subjects were divided into a coronary artery disease (CAD) group (n=70), a non-CAD group (n=61) and a control group (n=33). In the CAD group, 8-OHdG and biopyrrins levels increased with the severity of the New York Heart Association (NYHA) functional class and log BNP levels correlated with 8-OHdG and biopyrrins levels. However, non-CAD patients with NYHA class III/IV had significantly lower 8-OHdG levels than CAD patients with NYHA class III/IV and the levels did not correlate with log BNP levels. In the CAD group, 8-OHdG levels reflected the severity of atherosclerosis. These results indicate that the properties of oxidative stress markers should be carefully taken into consideration for the assessment of HF status.     

Assessment of oxidative DNA damage formation by organic complex mixtures from airborne particles PM(10)

The free radical generating activity of airborne particulate matter (PM₁₀) has been proposed as a primary mechanism in biological activity of ambient air pollution. In an effort to determine the impact of the complex mixtures of extractable organic matter (EOM) from airborne particles on oxidative damage to DNA, the level of 8-oxo-2′-deoxyguanosine (8-oxodG), the most prevalent and stable oxidative lesion, was measured in the human metabolically competent cell line Hep G2. Cultured cells were exposed to equivalent EOM concentrations (5–150 μg/ml) and oxidative DNA damage was analyzed using a modified single cell gel electrophoresis (SCGE), which involves the incubation of whole cell DNA with repair specific DNA endonuclease, which cleaves oxidized DNA at the sites of 8-oxodG. EOMs were extracted from PM₁₀ collected daily (24 h intervals) in three European cities: Prague (Czech Republic, two monitoring sites, Libuš and Smíchov), Košice (Slovak Republic) and Sofia (Bulgaria) during 3-month sampling periods in the winter and summer seasons. No substantial time- and dose-dependent increase of oxidative DNA lesions was detected in EOM-treated cells with the exception of the EOM collected at the monitoring site Košice, summer sampling. In this case, 2 h cell exposure to EOM resulted in a slight but significant increase of oxidative DNA damage at three from total of six concentrations. The mean 8-oxodG values at these concentrations ranged from 15.3 to 26.1 per 10⁶ nucleotides with a value 3.5 per 10⁶ nucleotides in untreated cells. B[a]P, the positive control, induced a variable but insignificant increase of oxidative DNA damage in Hep G2 cell (approximately 1.6-fold increase over control value).

Based on these data we believe that EOM samples extracted from airborne particle PM₁₀ play probably only a marginal role in oxidative stress generation and oxidative lesion formation to DNA. However, adsorbed organic compounds can undergo various interactions (additive or synergistic) with other PM components or physical factors (UV-A radiation) and in this way they might enhance/multiply the adverse health effects of air pollution.     

Hypertension aggravates glomerular dysfunction with oxidative stress in a rat model of diabetic nephropathy

Oxidative stress may contribute to the pathogenesis of diabetic nephropathy (DN), although the detailed mechanism of reactive oxygen species (ROS) regulation is still unclear. This study examined the effect of high-salt diet on ROS production and expression of antioxidant enzymes in control and experimentally diabetic rats. Wistar fatty rats (WFR) as a type 2 diabetes mellitus model and Wistar lean rats (WLR) as a control were fed a normal-salt diet (NS) and high-salt diet (HS) from the age of 6 to 14 weeks. We then examined the blood pressure, urinary albumin excretion (UAE), and urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels. The expression of antioxidant enzymes including α-catalase (CAT), Cu-Zn superoxide dismutase (SOD), Mn SOD, and glutathione peroxidase (GPx) were analyzed in the glomeruli of the rats using Western blotting. The expression of NAD(P)H oxidase p47^phox and NFκB p65 was evaluated using immunohistochemical staining. By 14 weeks of age, the WFR-HS group exhibited hypertension and markedly increased UAE. The level of 8-OHdG, a marker of oxidative damage, in the WFR-HS group was also higher than that in the WLR groups or WFR-NS group. The expression of α-CAT and Mn SOD proteins was significantly decreased in isolated glomeruli in the WFR-HS group. GPx and Cu-Zn SOD expression did not differ between the WFR and WLR groups. High expression of ROS and decreases in antioxidants were seen in the glomeruli of diabetic rats with hypertension, suggesting that oxidative stress may be involved in the development of DN.     

Protein expression in Down syndrome brain

Down syndrome (DS) is the most common chromosomal abnormality associated with early mental retardation and neurological abnormalities followed by precocious age dependent Alzheimer-type neurode generation later in life. Knowledge of the pathological mechanisms involved in DS is far from complete, but overexpression of genes residing in chromosome 21 was considered to be the central point for the DS phenotype. In this regard, beta amyloid precursor protein (APP), CuZn superoxide dismutase (SOD1) and S100beta have been implicated in causing apoptosis, a mechanism thought to be responsible for neuronal loss in DS, in one way or another. The gene dosage hypothesis has been challenged, however, and dysregulation of expression of genes located on other chromosomes has been described, which may well be secondary to chromosomal imbalance or a direct consequence of the disease process. The present review focuses on the protein expression profile in DS and we postulate that abnormalities in the coordinated expression, as well as interaction of proteins may be responsible for the neuropathology of DS. A series of candidate proteins are discussed that may be directly causing or reflecting the DS phenotype, in particular the brain abnormalities in DS.     

Ascorbate and H2O2 induced oxidative DNA damage in Jurkat cells

The effect of vitamin C (ascorbate) on oxidative DNA damage was examined by first incubating cells with dehydroascorbate, which boosts the intracellular concentration of ascorbate, and then exposing cells to H₂O₂. Oxidative DNA damage was estimated by the analysis of 5-hydroxy-2′-deoxycytidine (oh⁵dCyd) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxo⁸dGuo). The presence of a high concentration of ascorbate (30 mM), compared to the absence of ascorbate in cells, when exposed to H₂O₂ (200 μM), resulted in a remarkable sensitization of oh⁵dCyd from 2.7 ± 0.6 to 40.8 ± 6.1 lesions /10⁶ dCyd (15-fold). In contrast, the level of oxo⁸dGuo increased from 8.4 ± 0.4 to 12.1 ± 0.5 lesions/10⁶ dGuo (50%). The formation of oh⁵dCyd was also observed at lower concentrations of intracellular ascorbate and exogenous H₂O₂. Additional studies showed that replacement of H₂O₂ with tert-butyl hydroperoxide completely abolished damage, and that preincubation with iron and desferroxamine increased and decreased this damage, respectively. The latter studies suggest that a Fenton reaction is involved in the mechanism of damage. In conclusion, we report a novel model system in which ascorbate sensitizes H₂O₂-induced oxidative DNA damage in cells, leading to elevated levels of oh⁵dCyd and oxo⁸dGuo, with a strong bias toward the formation of oh⁵dCyd.     

Induction of 1,N(2)-etheno-2'-deoxyguanosine in DNA exposed to beta-carotene oxidation products

Epidemiological studies testing the effect of β-carotene in humans have found a relative risk for lung cancer in smokers supplemented with β-carotene. We investigated the reactions of retinal and β-apo-8′-carotenal, two β-carotene oxidation products, with 2′-deoxyguanosine to evaluate their DNA damaging potential. A known mutagenic adduct, 1,N²-etheno-2′-deoxyguanosine, was isolated and characterized on the basis of its spectroscopic features. After treatment of calf thymus DNA with β-carotene or β-carotene oxidation products, significantly increased levels of 1,N²-etheno-2′-deoxyguanosine and 8-oxo-7,8-dihydro-2′-deoxyguanosine were quantified in DNA. These lesions are believed to be important in the development of human cancers. The results reported here may contribute toward an understanding of the biological effects of β-carotene oxidation products.