Oxidative stress in the alcoholic liver disease

Parameters reflecting oxidative stress (OS) have been studied in 37 patients with alcoholic liver disease (ALD) during admission to the hospital and 2 weeks after the beginning of therapy. The patients were divided into 3 groups: alcoholic hepatitis (AH), alcoholic cirrhosis with hepatic insufficiency (the group C by the Child-Paquet scale) and terminal stage patients (subsequently died). All patients were characterized by a significant increase in plasma products of lipid peroxidation (conjugated diene and malondialdehyde) and a decrease of the ceruloplasmin level. The coefficient C OS significantly exceeded normal values both on admission and after the 2-week course of traditional therapy. This suggests an important role of the OS with ALD.     

Oxidative and non-oxidative DNA damage and cardiovascular disease

Abstract

Evidence for the association of DNA damage with cardiovascular disease has been obtained from in vitro cell culture models, experimental cardiovascular disease and analysis of samples obtained from humans with disease. There is general acceptance that several factors associated with the risk of developing cardiovascular disease cause oxidative damage to DNA in cell culture models with both nuclear and mitochondrial DNA as targets. Moreover, evidence obtained over the past 10 years points to a possible mechanistic role for DNA damage in experimental atherosclerosis culminating in recent studies challenging the assumption that DNA damage is merely a biomarker of the disease process. This kind of mechanistic insight provides a renewed impetus for further studies in this area.     

Oxidative and non-oxidative DNA damage and cardiovascular disease

Evidence for the association of DNA damage with cardiovascular disease has been obtained from in vitro cell culture models, experimental cardiovascular disease and analysis of samples obtained from humans with disease. There is general acceptance that several factors associated with the risk of developing cardiovascular disease cause oxidative damage to DNA in cell culture models with both nuclear and mitochondrial DNA as targets. Moreover, evidence obtained over the past 10 years points to a possible mechanistic role for DNA damage in experimental atherosclerosis culminating in recent studies challenging the assumption that DNA damage is merely a biomarker of the disease process. This kind of mechanistic insight provides a renewed impetus for further studies in this area.     

Oxidative mechanisms in the pathogenesis of alcoholic liver disease

Although the capacity of ethanol to induce oxidative stress in the liver is well established, the mechanisms by which oxidative damage contributes to the pathogenesis of alcoholic liver disease (ALD) is still incompletely understood. Recent reports have implicated oxidative mechanisms in the onset of alcoholic steatosis and in the formation of Mallory's bodies. Moreover, by inducing mitochondrial alterations, oxidative stress promotes hepatocyte necrosis and contributes to alcohol-induced sensitization of hepatocyte to the pro-apoptotic action of TNF-alpha. Oxidative mechanisms play also a role in the progression of liver fibrosis by triggering the release of pro-fibrotic cytokines and activating collagen gene expression in hepatic stellate cells. Finally, immune responses towards antigens originating from the reactions of lipid peroxidation products with hepatic proteins might represent one of the mechanisms that contribute to perpetuate chronic hepatic inflammation in ALD. Altogether these observations give a rationale to the possible clinical application of antioxidants in the therapy of ALD.     

Oxidative DNA damage and DNA repair enzyme expression are inversely related in murine models of fatty liver disease

Mitochondrial generation of reactive oxygen species (ROS) is increased in mice with fatty livers induced by genetic obesity, chronic consumption of ethanol, or methionine/choline-deficient diets. Both nuclear and mitochondrial (mt) DNA are targets for ROS-induced damage and accumulate hydroxylated bases, such as 8-hydroxy-2'-deoxyguanosine (8-oxoG) and base substitution of adenine with 8-oxoG (A*8-oxoG), that introduce mutations that promote cancer as well as cell death. The mammalian homolog of the bacterial DNA mismatch repair enzyme MutY (MYH) removes A*8-oxoG from nuclear and mtDNA, reduces 8-oxoG accumulation, and restores genomic stability after ROS exposure. Cumulative damage to mtDNA occurs as fatty liver disease progresses. Therefore, differences in hepatic MYH activity may influence the severity of fatty liver disease. To evaluate this hypothesis, we compared mtH2O2 production, MYH expression, oxidative DNA damage, and hepatocyte death in healthy mice and different mouse models of fatty liver disease. The results show that diverse causes of steatohepatitis increase mtROS production, limit repair of mtDNA, and oxidatively damage DNA. However, there are important differences in the DNA repair response to oxidant stress among mouse models of fatty liver disease. Independent of the degree of mtROS generation, models with the least MYH exhibit the greatest accumulation of 8-oxoG and the most hepatocyte death. These findings raise the intriguing possibility that inherited or acquired differences in DNA repair enzyme activity may underlie some of the interindividual differences in fatty liver disease outcomes.     

Oxidative damage in Parkinson disease: Measurement using accurate biomarkers

Oxidative damage has been implicated in the pathogenesis of Parkinson disease (PD) but the literature data are confusing. Using products of lipid and DNA oxidation measured by accurate methods, we assessed the extent of oxidative damage in PD patients. The levels of plasma F₂-isoprostanes (F₂-IsoPs), hydroxyeicosatetraenoic acid products (HETEs), cholesterol oxidation products, neuroprostanes (F₄-NPs), phospholipase A₂ (PLA₂) and platelet activating factor–acetylhydrolase (PAF-AH) activities, urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG), and serum high-sensitivity C-reactive protein were compared in 61 PD patients and 61 age-matched controls. The levels of plasma F₂-IsoPs, HETEs, 7β-and 27-hydroxycholesterol, 7-ketocholesterol, F₄-NPs, and urinary 8-OHdG were elevated, whereas the levels of plasma PLA₂ and PAF-AH activities were lower, in PD patients compared to controls (p <  0.05). The levels of plasma F₂-IsoPs, HETEs, and urinary 8-OHdG were higher in the early stages of PD (p trend <  0.05). There was a significant negative correlation between the cumulative intake of levodopa and urinary 8-OHdG (r =  ?0.305, p =  0.023) and plasma total HETEs (r =  ?0.285, p =  0.043). Oxidative damage markers are systemically elevated in PD, which may give clues about the relation of oxidative damage to the onset and progression of PD.     

Oxidative DNA damage in mild cognitive impairment and late-stage Alzheimer's disease

Increasing evidence supports a role for oxidative DNA damage in aging and several neurodegenerative diseases including Alzheimer's disease (AD). Attack of DNA by reactive oxygen species (ROS), particularly hydroxyl radicals, can lead to strand breaks, DNA–DNA and DNA–protein cross-linking, and formation of at least 20 modified bases adducts. In addition, α,β-unsaturated aldehydic by-products of lipid peroxidation including 4-hydroxynonenal and acrolein can interact with DNA bases leading to the formation of bulky exocyclic adducts. Modification of DNA bases by direct interaction with ROS or aldehydes can lead to mutations and altered protein synthesis. Several studies of DNA base adducts in late-stage AD (LAD) brain show elevations of 8-hydroxyguanine (8-OHG), 8-hydroxyadenine (8-OHA), 5-hydroxycytosine (5-OHC), and 5-hydroxyuracil, a chemical degradation product of cytosine, in both nuclear and mitochondrial DNA (mtDNA) isolated from vulnerable regions of LAD brain compared to age-matched normal control subjects. Previous studies also show elevations of acrolein/guanine adducts in the hippocampus of LAD subjects compared to age-matched controls. In addition, studies of base excision repair show a decline in repair of 8-OHG in vulnerable regions of LAD brain. Our recent studies show elevated 8-OHG, 8-OHA, and 5,6-diamino-5-formamidopyrimidine in both nuclear and mtDNA isolated from vulnerable brain regions in amnestic mild cognitive impairment, the earliest clinical manifestation of AD, suggesting that oxidative DNA damage is an early event in AD and is not merely a secondary phenomenon.     

Oxidative damage in cultured human olfactory neurons from Alzheimer's disease patients

Oxidative abnormalities precede clinical and pathological manifestations of Alzheimer's disease and are the earliest pathological changes reported in the disease. The olfactory pathways and mucosa also display the pathological features associated with Alzheimer's disease in the brain. Olfactory neurons are unique because they can undergo neurogenesis and are able to be readily maintained in cell culture. In this study, we examined neuronal cell cultures derived from olfactory mucosa of Alzheimer's disease and control patients for oxidative stress responses. Levels of lipid peroxidation (hydroxynonenal), N(epsilon)-(carboxymethyl)lysine (glycoxidative and lipid peroxidation), and oxidative stress response (heme oxygenase-1) were measured immunocytochemically. We found increased levels for all the oxidative stress markers examined in Alzheimer's disease neurons as compared to controls. Interestingly, in one case of Alzheimer's disease, we found hydroxynonenal adducts accumulated in cytoplasmic lysosome-like structures in about 20% of neurons cultured, but not in neurons from control patients. These lysosome-like structures are found in about 100% of the vulnerable neurons in brains of cases of Alzheimer's disease. This study suggests that manifestations of oxidative imbalance in Alzheimer's disease extend to cultured olfactory neurons. Primary culture of human olfactory neurons will be useful in understanding the mechanism of oxidative damage in Alzheimer's disease and can even be utilized in developing therapeutic strategies.     

Oxidative modifications impair albumin quantification

Background

Hypoalbuminemia is a measure of malnutrition, inflammation and a predictor of mortality in uremia. It is controversial whether albumin levels per se are associated with the clinical outcomes in uremic patients. The co-occurrence of hypoalbuminemia and oxidative stress in hemodialysis (HD) patients led us to hypothesize that oxidative modifications of albumin decrease its detection and influence albumin quantification.

Methods

Albumin levels are determined in clinical laboratories mainly by the bromocresol green (BCG) spectrophotometric assay. The detection of serum albumin was investigated in HD patients and in healthy controls using an “albumin-detection index”, defined as the ratio between BCG read-out (albumin-specific) to total albumin. The detection efficacy of albumin was also investigated in vitro, after glycoxidation, HOCl-mediated-oxidation, and metal-catalyzed-oxidation. Oncotic pressure was measured to assess albumin function.

Results

The albumin-detection index of patients was significantly lower compared with controls, correlating negatively with oxidative stress markers (serum advanced oxidation protein products-AOPP and glycoxidized serum albumin) and positively with serum albumin levels. The albumin-detection index was also decreased after in vitro oxidation.

Conclusions

The study shows, both in vivo and in vitro, decreased detection of oxidized albumin by a commonly-used clinical assay, thus providing the molecular link between oxidative stress and hypoalbuminemia. Oxidative stress as reflected by hypoalbuminemia, rather than actual albumin levels, may be related to cardiovascular morbidity outcomes in HD patient.     

Oxidative damage in liver after perinatal intoxication with lead and/or cadmium

Lead acetate (300 mg Pb/L) and/or cadmium acetate (10mg Cd/L) in blood and liver were administrated as drinking water to pregnant Wistar rats from day 1 of pregnancy to parturition (day 0) or until weaning (day 21), to investigate the toxic effects in blood and in the liver. Both metals produced mycrocitic anaemia in the pups as well as oxidative damage in the liver, as suggested by the significant increase in TBARS production and the high catalase activity. Moreover, intense alkaline and acid phosphatase activity, used as biomarkers of liver adaptation to damaging factors, was observed. In addition, the toxikinetics are different for Pb and Cd: while Cd is a hepatotoxic from day 0, Pb is not until day 21. Finally, simultaneous perinatal administration of both metals seems to protect, at least, in the liver TBARS production against the toxicity produced by Cd or Pb separately.     

Oxidative damage to macromolecules in human Parkinson disease and the rotenone model

Parkinson disease (PD), the most common neurodegenerative movement disorder, is associated with selective degeneration of nigrostriatal dopamine neurons. Although the underlying mechanisms contributing to neurodegeneration in PD seem to be multifactorial, mitochondrial impairment and oxidative stress are widely considered to be central to many forms of the disease. Whether oxidative stress is a cause or a consequence of dopaminergic death, there is substantial evidence for oxidative stress both in human PD patients and in animal models of PD, especially using rotenone, a complex I inhibitor. There are many indices of oxidative stress, but this review covers the recent evidence for oxidative damage to nucleic acids, lipids, and proteins in both the brain and the peripheral tissues in human PD and in the rotenone model. Limitations of the existing literature and future perspectives are discussed. Understanding how each particular macromolecule is damaged by oxidative stress and the interplay of secondary damage to other biomolecules may help us design better targets for the treatment of PD.     

Gallstone disease is associated with more severe liver damage in patients with non-alcoholic Fatty liver disease

Background

Nonalcoholic fatty liver disease (NAFLD) and gallstone disease (GD) are both highly prevalent in the general population and associated with obesity and insulin resistance. We aimed to evaluate the prevalence of GD in a cross sectional study of NAFLD patients and to define whether the presence of GD is associated with diabetes and predicts more severe liver disease.

Methodology/Principal Findings

We merged databases of four Liver Units, comprising 524 consecutive biopsy-proven NAFLD (373 males) observed between January 2003 and June 2010. GD was diagnosed in 108 (20%), and 313 cases (60%) were classified by liver biopsy as nonalcoholic steatohepatitis (NASH). The GD subgroup was characterized by a significantly higher prevalence of females, prediabetes/diabetes, abdominal obesity and metabolic syndrome, older age, higher BMI, fasting glucose, HOMA-IR and lower ALT. The prevalence of GD progressively increased with advancing fibrosis and with the severity of necroinflammatory activity (p for trend  = 0.0001 and  = 0.01, respectively), without differences in the severity of steatosis. At multivariate analysis GD was associated with female gender (OR 1.37, 95% CI 1.04–1.8), age (OR 1.027, 95% CI1.003–1.05), fasting glucose (OR 1.21, 95% CI 1.10–1.33) and NASH (OR 1.40,95% CI 1.06–1.89), whereas ALT levels were associated with a lower GD risk (OR 0.98, 95% CI 0.97–0.99). When subjects with cirrhosis were excluded from analysis, the association between GD and fasting glucose, female gender, and NASH was maintained.

Conclusion

Patients with NAFLD have a high prevalence of GD, which characterizes subjects with altered glucose regulation and more advanced liver disease.     

Oxidative and alkylating damage in DNA

Modification of cellular DNA upon exposure to reactive oxygen and nitrogen species is the likely initial event involved in the induction of the mutagenic and lethal effects of various oxidative stress agents. Evidence has been accumulated for the significant implication of singlet oxygen (1O(2)), generated as the result of UVA activation of endogenous photosensitizers as porphyrins and flavins. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) has been shown to be the exclusive product of the reaction of 1O(2) with the guanine moiety of cellular DNA, in contrast to the hydroxyl radical, which reacts almost indifferently with all the nucleobases and the sugar moiety of DNA. Furthermore 8-oxodGuo is also produced by other oxidants and can be used as an ubiquitous biomarker of DNA oxidation but can not be a specific marker of any particular species. The role of DNA etheno adducts in mutagenic and carcinogenic processes triggered by known occupational and environmental carcinogens has also been studied. Much interest in etheno adducts resulted from the detection of increased levels of 1,N(6)-etheno-2'-deoxyadenosine and 3,N(4)-etheno-2'-deoxycytidine in DNA from human, rat and mouse tissues under pathophysiological conditions associated with oxidative stress. A method involving on-line HPLC with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) in DNA. This methodology permits direct quantification of 20 fmol (7.4 adducts/10(8) dGuo) of the etheno adduct from approximately 350 microg of crude DNA hydrolysates. This method provides the first evidence of the occurrence of 1,N(2)-epsilondGuo as a basal endogenous lesion and may be utilized to better assess the biological consequences of etheno DNA damage under normal and pathological conditions. This work addresses the importance of isotope labeling associated with mass spectrometry technique for biomolecule damage studies.     

Oxidative damage in age-related macular degeneration

Epidemiologic studies have suggested that elderly patients who consumed diets rich in antioxidants throughout their lives are less likely to be afflicted with age-related macular degeneration (AMD). This led to the Age-Related Eye Disease Study, which showed that supplements containing antioxidant vitamins and zinc reduce the risk of progression to severe stages of AMD. Despite these data that indirectly implicate oxidative damage in the pathogenesis of AMD, there has not been any direct demonstration of increased oxidative damage in the retinas of patients with AMD. In this study, we used biomarkers of oxidative damage in postmortem eyes from patients with AMD and comparably aged patients without AMD to directly assess for oxidative damage. Sections from 4 eyes with no pathologic features of AMD showed no immunofluorescent staining for markers of oxidative damage, while sections from 8 of 12 eyes with advanced geographic atrophy showed evidence of widespread oxidative damage in both posterior and anterior retina. Only 2 of 8 eyes with choroidal neovascularization and 2 of 16 eyes with diffuse drusen and no other signs of AMD showed evidence of oxidative damage. These data suggest that widespread oxidative damage occurs in the retina of some patients with AMD and is more likely to be seen in patients with advanced geographic atrophy. This does not rule out oxidative damage as a pathogenic mechanism in patients with CNV, but suggests that a subpopulation of patients with geographic atrophy may have a major deficiency in the oxidative defense system that puts the majority of cells in the retina at risk for oxidative damage.     

Oxidative modification of albumin in the parenchymal lung tissue of current smokers with chronic obstructive pulmonary disease

Background

There is accumulating evidence that oxidative stress plays an important role in the pathophysiology of chronic obstructive pulmonary disease (COPD). One current hypothesis is that the increased oxidant burden in these patients is not adequately counterbalanced by the lung antioxidant systems.

Objective

To determine the levels of oxidised human serum albumin (HSA) in COPD lung explants and the effect of oxidation on HSA degradation using an ex vivo lung explant model.

Methods

Parenchymal lung tissue was obtained from 38 patients (15F/23M) undergoing lung resection and stratified by smoking history and disease using the GOLD guidelines and the lower limit of normal for FEV₁/FVC ratio. Lung tissue was homogenised and analysed by ELISA for total levels of HSA and carbonylated HSA. To determine oxidised HSA degradation lung tissue explants were incubated with either 200 μg/ml HSA or oxidised HSA and supernatants collected at 1, 2, 4, 6, and 24 h and analysed for HSA using ELISA and immunoblot.

Results

When stratified by disease, lung tissue from GOLD II (median = 38.2 μg/ml) and GOLD I (median = 48.4 μg/ml) patients had lower levels of HSA compared to patients with normal lung function (median = 71.9 μg/ml, P < 0.05). In addition the number of carbonyl residues, which is a measure of oxidation was elevated in GOLD I and II tissue compared to individuals with normal lung function (P < 0.05). When analysing smoking status current smokers had lower levels of HSA (median = 43.3 μg/ml, P < 0.05) compared to ex smokers (median = 71.9 μg/ml) and non-smokers (median = 71.2 μg/ml) and significantly greater number of carbonyl residues per HSA molecule (P < 0.05). When incubated with either HSA or oxidised HSA lung tissue explants rapidly degraded the oxidised HSA but not unmodified HSA (P < 0.05).

Conclusion

We report on a reliable methodology for measuring levels of oxidised HSA in human lung tissue and cell culture supernatant. We propose that differences in the levels of oxidised HSA within lung tissue from COPD patients and current smokers provides further evidence for an oxidant/antioxidant imbalance and has important biological implications for the disease.

     

Hepatic protoporphyrin metabolism in patients with advanced protoporphyric liver disease.

Protoporphyria is a genetic disorder in which liver damage is caused by the toxic effect of protoporphyrin accumulation in the liver. In this study protoporphyrin was measured in the resected livers of 7 patients who had liver transplantation and an additional patient from whom liver tissue was obtained post mortem. Comparison of liver, erythrocyte and serum protoporphyrin levels demonstrated a marked gradient between these compartments: erythrocyte, 5781 +/- 655 micrograms/dl; serum, 384 +/- 102 micrograms/dl; liver 377,238 +/- 55,568 micrograms/100 gm wet weight, (mean +/- SE). Protoporphyrin levels in bile of 3 patients were 55,559, and 1,153 micrograms/dl, indicating a gradient between liver and bile as well. Examination of the livers by polarization microscopy and electron microscopy demonstrated protoporphyrin pigment crystals. In one patient who had recurrent liver disease after transplantation, the protoporphyrin concentration in the graft at the time of death was similar to that in the resected liver. These data indicate that liver protoporphyrin levels in patients with advanced protoporphyric liver disease are much higher than levels in blood and bile, in part because protoporphyrin forms crystalline deposits in liver tissue. Thus, progressive hepatic accumulation of protoporphyrin occurs in the face of impaired biliary excretion. An intrinsic defect in hepatic excretion of protoporphyrin is probably not necessary for this condition to develop because liver disease can occur in the graft following transplantation.