Base excision DNA repair levels in mitochondrial lysates of Alzheimer's disease

Alzheimer's disease (AD) is a senile dementia with increased incidence in older subjects (age >65 years). One of the earliest markers of AD is oxidative DNA damage. Recently, it has been reported that preclinical AD patient brains show elevated levels of oxidative damage in both nuclear and mitochondrial nucleic acids. Moreover, different oxidative lesions in mitochondrial DNA are between 5- and 10-fold higher than in nuclear DNA in both control and AD postmortem brains. We previously showed that there is a significant loss of base excision repair (BER) components in whole tissue extracts of AD and mild cognitive impairment subjects relative to matched control subjects. However, comprehensive analysis of specific steps in BER levels in mitochondrial extracts of AD patient brains is not available. In this study, we mainly investigated various components of BER in mitochondrial extracts of AD and matched control postmortem brain samples. We found that the 5-hydroxyuracil incision and ligase activities are significantly lower in AD brains, whereas the uracil incision, abasic site cleavage, and deoxyribonucleotide triphosphate incorporation activities are normal in these samples.     

Prognostic impact of 8‐hydroxy‐deoxyguanosine and its repair enzyme 8‐hydroxy‐deoxyguanosine DNA glycosylase in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has a poor prognosis in the setting of chronic inflammation and fibrosis, both of which promote nuclear DNA oxidative damage. 8‐hydroxy‐deoxyguanosine (8‐OHdG) DNA glycosylase (OGG1) enhances the repair of 8‐OHdG, which is the primary oxidative stress‐induced mutation that leads to malignant alterations. This study aims to clarify the relationships between oxidative stress‐induced factors and HCC progression. The clinicopathological factors were compared with immunohistochemistry OGG1 and 8‐OHdG expressions in 86 resected HCC specimens. High 8‐OHdG expression was associated with high serum aspartate transaminase and total bilirubin levels, as well as a low platelet count, compared with low 8‐OHdG expression. Histological liver cirrhosis and poor differentiation were more frequent in patients with high 8‐OHdG expression than in those with low 8‐OHdG expression. The 8‐OHdG was negatively correlated with OGG1 expression in HCC patients. Therefore, we classified the patients into two groups, low OGG1/high 8‐OHdG group and the other group. The patients with low OGG1/high 8‐OHdG expressions had worse prognosis than those with the other expressions. Our results showed that low OGG1/high 8‐OHdG expressions in nuclei influence HCC patient outcomes. Evaluating the patterns of OGG1 and 8‐OHdG expressions might provide pivotal prognostic biomarkers in patients with HCC.     

Elevated levels of oxidative DNA damage in lymphocytes from patients with Alzheimer's disease.

Previous studies have provided evidence of the involvement of oxidative damage in the pathogenesis of Alzheimer's disease (AD). Although the role of oxidative stress in the aetiology of the disease is still not clear, the detection of an increased damage status in the cells of patients could have important therapeutic implications. The level of oxidative damage and repair capacity in peripheral lymphocytes of AD patients and of age-matched controls was determined by the Comet assay applied to freshly isolated blood samples with oxidative lesion-specific DNA repair endonucleases. This is less prone to errors arising from oxidative artifacts than chemical analytical methods; and is therefore a relatively reliable, as well as rapid method for assay of oxidative DNA damage in cells. Statistically significant elevations (P < 0.05) of oxidized purines were observed in nuclear DNA of peripheral lymphocytes from AD patients, compared to age matched control subjects, both at basal level and after oxidative stress induced by H(2)O(2.) AD patients also showed a diminished repair of H(2)O(2) -induced oxidized purines.     

A Ser326Cys polymorphism in the DNA repair gene hOGG1 is not associated with sporadic Alzheimer's disease

Oxidative damage accumulates in the DNA of the human brain over time, and is supposed to play a critical role in the pathogenesis of Alzheimer's disease (AD). It has been suggested that the brain in AD might be subjected to the double insult of increased oxidative stress, as well as deficiencies in repair mechanisms responsible for the removal of oxidized bases. The type of damage that is most likely to occur in neuronal cells is oxidative DNA damage which is primarily removed by the base excision repair (BER) pathway, and a decrease in BER activity was observed in post-mortem brain regions of AD individuals, especially in the activity of 8-oxoguanine DNA glycosylase. There is evidence that the Ser326Cys polymorphism of the human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene is associated with a reduced DNA repair activity. However, although a deficient BER was proposed in the etiology of AD by several authors, polymorphisms of BER genes have not been studied in AD yet. We performed a case-control study including 178 patients with sporadic AD (sAD) and 146 matched controls to evaluate the role of the Ser326Cys polymorphism as a risk factor for sAD. In the present study we failed to find any association between allele (chi2=0.03, p=0.86) or genotype (chi2=0.25, p=0.882) frequencies of hOGG1 Ser326Cys and the risk of sAD. Present results suggest that the Ser326Cys polymorphism of the hOGG1 gene is not an independent risk factor for sAD.     

Oxidative Alterations in Alzheimer's Disease

There is increasing evidence that free radical damage to brain lipids, carbohydrates, proteins, and DNA is involved in neuron death in neurodegenerative disorders. The largest number of studies have been performed in Alzheimer's disease (AD) where there is considerable support for the oxidative stress hypothesis in the pathogenesis of neuron degeneration. In autopsied brain there is an increase in lipid peroxidation, a decline in polyunsaturated fatty acids (PUFA) and an increase in 4-hydroxynonenal (HNE), a neurotoxic aldehyde product of PUFA oxidation. Increased protein oxidation and a marked decline in oxidative-sensitive enzymes, glutamine synthetase and creatinine kinase, are found in the brain in AD. Increased DNA oxidation, especially 8-hydroxy-2'-deoxyguanosine (8-OHdG) is present in the brain in AD. Immunohistochemical studies show the presence of oxidative stress products in neurofibrillary tangles and senile plaques in AD. Markers of lipid peroxidation (HNE, isoprostanes) and DNA (8-OHdG) are increased in CSF in AD. In addition, inflammatory response markers (the complement cascade, cytokines, acute phase reactants and proteases) are present in the brain in AD. These findings, coupled with epidemiologic studies showing that anti-inflammatory agents slow the progression or delay the onset of AD, suggest that inflammation plays a role in AD. Overall these studies indicate that oxidative stress and the inflammatory cascade, working in concert, are important in the pathogenetic cascade of neurodegeneration in AD, suggesting that therapeutic efforts aimed at both of these mechanisms may be beneficial.     

Oxidative damages in chronic inflammation of a mouse autoimmune disease model

Reactive oxygen species are generated in many types of inflammation; it is unclear, however, if inflammation leads to oxidative damage of DNA, proteins and lipids within the inflamed tissues. In this study, we used mice that are homozygous for the alymphoplasia (aly) mutation as a model to determine if inflammation induces oxidative damage in liver and pancreas. We found that 8-hydroxy-2'-deoxyguanosine (8OHdG), which is a product of oxidative DNA damage, increases with age in livers and pancreata of C57BL/6aly/aly (aly/aly) and C57BL/6 wild type (WT) mice. The 8OHdG levels in liver, but not in pancreas, of aged aly/aly mice were significantly higher than those in age-matched WT mice. We showed that aging enhances oxidative protein damage, as measured by carbonylated protein contents, in the pancreata of WT but not aly/aly mice. In contrast, neither aging nor inflammation was associated with lipid damage, as measured by thiobarbituric acid-reactive substances (TBARS), in aly/aly or WT mice. Our results indicate that chronic inflammation in liver but not pancreas leads to increased oxidative damage to DNA, but not to lipids and proteins in aly/aly mouse model.     

Increased levels of DNA breaks in cerebral cortex of Alzheimer's disease patients

It has been hypothesized that Alzheimer's disease (AD) is caused by an accumulation of damage in DNA due to defective DNA-repair (21). Attempts to test this hypothesis by determining the activity of DNA-repair systems in nonneuronal cells from AD patients and controls so far provided conflicting results. An alternative approach is the direct comparison of DNA-damage levels in neuronal tissue of AD patients and controls. In the present study we assayed the level of DNA breaks and alkali-labile sites in cerebral cortex tissue samples from AD patients and controls obtained from rapid autopsies. Our data on 11 AD patients and 8 control subjects indicate an at least two-fold higher level of DNA damage in cortex of AD patients as compared to controls.     

Morphological and biochemical assessment of DNA damage and apoptosis in Down syndrome and Alzheimer disease, and effect of postmortem tissue archival on TUNEL

We have previously shown that Alzheimer disease (AD) brain exhibits terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) for DNA damage and morphological evidence for apoptosis. Down syndrome (DS) is a neurodegenerative disorder that exhibits significant neuropathological parallels with AD. In accordance with these parallels and the need to clarify the mechanism of cell death in DS and AD, we investigated two principal issues in the present study. First, we investigated the hypothesis that TUNEL labeling for DNA damage and morphological evidence for apoptosis is also present in the DS brain. All DS cases employed had a neuropathological diagnosis of AD. Analysis of these cases showed that DS brain exhibits a significant increase in the number of TUNEL-labeled nuclei relative to controls matched for age, Postmortem Delay, and Archival Length, and that a subset of TUNEL-positive nuclei exhibits apoptotic morphologies. We also report that Archival Length in 10% formalin can significantly affect TUNEL labeling in postmortem human brain, and therefore, that Archival Length must be controlled for as a variable in this type of study. Second, we investigated whether biochemical evidence for the mechanism of cell death in DS and AD could be detected. To address this question we employed pulsed-field gel electrophoresis (PFGE) as a sensitive method to evaluate DNA integrity. Although apoptotic oligonucleosomal laddering has not previously been observed in AD, PFGE of DNA from control, DS and AD brain in the present study revealed evidence of high molecular weight DNA fragmentation indicative of apoptosis. This represents biochemical support for an apoptotic mechanism of cell death in DS and AD.     

A longitudinal study of atrazine and 2,4‐D exposure and oxidative stress markers among iowa corn farmers

Reactive oxygen species, potentially formed through environmental exposures, can overwhelm an organism's antioxidant capabilities resulting in oxidative stress. Long‐term oxidative stress is linked with chronic diseases. Pesticide exposures have been shown to cause oxidative stress in vivo. We utilized a longitudinal study of corn farmers and non‐farming controls in Iowa to examine the impact of exposure to the widely used herbicides atrazine and 2,4‐dichlorophenoxyacetic acid (2,4‐D) on markers of oxidative stress. 225 urine samples were collected during five agricultural time periods (pre‐planting, planting, growing, harvest, off‐season) for 30 farmers who applied pesticides occupationally and 10 controls who did not; all were non‐smoking men ages 40–60. Atrazine mercapturate (atrazine metabolite), 2,4‐D, and oxidative stress markers (malondialdehyde [MDA], 8‐hydroxy‐2′‐deoxyguanosine [8‐OHdG], and 8‐isoprostaglandin‐F_2α [8‐isoPGF]) were measured in urine. We calculated β estimates and 95% confidence intervals (95%CI) for each pesticide‐oxidative stress marker combination using multivariate linear mixed‐effect models for repeated measures. Farmers had higher urinary atrazine mercapturate and 2,4‐D levels compared with controls. In regression models, after natural log transformation, 2,4‐D was associated with elevated levels of 8‐OHdG (β = 0.066, 95%CI = 0.008–0.124) and 8‐isoPGF (β = 0.088, 95%CI = 0.004–0.172). 2,4‐D may be associated with oxidative stress because of modest increases in 8‐OHdG, a marker of oxidative DNA damage, and 8‐isoPGF, a product of lipoprotein peroxidation, with recent 2,4‐D exposure. Future studies should investigate the role of 2,4‐D‐induced oxidative stress in the pathogenesis of human diseases. Environ. Mol. Mutagen. 58:30–38, 2017. © 2016 Wiley Periodicals, Inc.     

3-Nitrotyrosine immunoreactivity in olfactory receptor neurons of patients with Alzheimer's disease: implications for impaired odor sensitivity

Olfactory sensory function is impaired in patients with the diagnosis of probable Alzheimer's disease (AD) compared to elderly controls, and the olfactory epithelium (OE) of AD patients exhibits several pathological changes characteristic of the AD brain. To confirm that the populations from whom our postmortem tissues are obtained exhibit similar decrements in sensory function, threshold testing was performed; probable AD patients had significantly higher olfactory thresholds than controls. To determine if oxidative stress contributes to decreased olfactory function in AD, we localized 3-nitrotyrosine (3-NT) immunoreactivity in OE obtained postmortem from patients with neuropathologically confirmed AD and age-matched controls with brains free of significant neurodegenerative pathology. In AD patients, immunoreactivity was localized in olfactory receptor neurons (ORNs), including dendritic knobs where ion channels that participate in sensory transduction are located, suggesting a direct mechanism for olfactory impairment. In controls, immunoreactivity occurred in blood vessel endothelium, suggesting age-related vascular dysfunction. Immunohistochemistry for CD68, a macrophage scavenger receptor, demonstrated activated macrophages, a source of free radicals contributing to 3-NT formation, in the OE of AD patients but not controls. These results demonstrate increased oxidative stress and modification of ORN proteins that may contribute directly to olfactory impairment in AD patients.     

DNA oxidation in Alzheimer's disease

Oxidative damage to DNA may play an important role in aging and neurodegenerative diseases such as Alzheimer's disease (AD). Attack on DNA by reactive oxygen species, particularly hydroxyl radicals, can lead to strand breaks, DNA-DNA and DNA-protein cross-linking, sister chromatid exchange and translocation, and formation of at least 20 oxidized base adducts. Modification of DNA bases can lead to mutation and altered protein synthesis. In late-stage AD brain, several studies have shown an elevation of the base adducts 8 hydroxyguanine (8-OHG), 8-hydroxyadenine (8-OHA), 5-hydroxycytosine (5-OHC), and 5-hydroxyuracil, a chemical degradation product of cytosine. Several studies have shown a decline in repair of 8-OHG in AD. Most recently, our studies have shown elevated 8-OHG, 8-OHA, and 5,6-diamino-5-formamidopyrimidine in nuclear and mitochondrial DNA in mild cognitive impairment, the earliest detectable form of AD, suggesting that oxidative damage to DNA is an early event in AD and not a secondary phenomenon.     

Heme-a, the heme prosthetic group of cytochrome c oxidase,is increased in Alzheimer's disease

Heme-a, is the heme prosthetic group of cytochrome c oxidase (COX), the terminal complex of the mitochondrial electron transport chain. We measured heme-a levels in postmortem brain tissue from nine patients diagnosed with dementia: Alzheimer's disease (AD) was the primary diagnosis in five, AD/diffuse Lewy body disease (DLBD) was diagnosed in two, DLBD was diagnosed in one, and DLBD (severe)/AD (mild) was diagnosed in one. Eight non-demented patients who died from non-neurological causes served as controls. When the primary diagnosis was AD (AD and AD/DLBD), levels of cerebral heme-a were increased almost two-fold on a protein basis compared to controls (p < 0.001). Using perfused and non-perfused rats we showed that measured levels of cerebral heme-a were unaffected by the presence of blood in brain tissue. In mice we showed that levels of cerebral heme-a were unaffected by 24 h of storage at 4 °C prior to freezing. These animal studies suggest that increased levels of cerebral heme-a in AD were not due to blood in postmortem brain or variation in postmortem interval.     

Mitochondrial DNA damage as a mechanism of cell loss in Alzheimer's disease

Aging is associated with impaired mitochondrial function caused by accumulation of oxygen free radical-induced mitochondrial (Mt) DNA mutations. One prevailing theory is that age-associated diseases, including Alzheimer's disease (AD), may be precipitated, propagated, or caused by impaired mitochondrial function. To investigate the role of MtDNA relative to genomic (Gn) DNA damage in AD, temporal lobe samples from postmortem AD (n = 37) and control (n = 25) brains were analyzed for MtDNA and GnDNA fragmentation, mitochondrial protein and cytochrome oxidase expression, MitoTracker Green fluorescence (to assess mitochondrial mass/abundance), and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG) immunoreactivity. Brains with AD had more extensive nicking and fragmentation of both MtDNA and GnDNA as demonstrated by agarose gel electrophoresis, end-labeling, and the in situ terminal deoxynucleotide transferase end-labeling (TUNEL) assay, and only the brains with AD had detectable 8-OHdG immunoreactivity in cortical neurons. Increased MtDNA damage in AD was associated with reduced MtDNA content, as demonstrated by semiquantitative PCR analysis and reduced levels of Mt protein and cytochrome oxidase expression by Western blot analysis or immunohistochemical staining with image analysis. The finding of reduced MitoTracker Green fluorescence in AD brains provided additional evidence that reduced Mt mass/abundance occurs with AD neurodegeneration. The presence of increased MtDNA and GnDNA damage in AD suggest dual cell death cascades in AD. Impaired mitochondrial function caused by MtDNA damage may render brain cells in AD more susceptible to oxidative injury and thereby provide a mechanism by which systemic or environmental factors could influence the course of disease.     

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.     

Global changes in DNA methylation and hydroxymethylation in Alzheimer's disease human brain

DNA methylation (5-methylcytosine [5mC]) is one of several epigenetic markers altered in Alzheimer's disease (AD) brain. More recently, attention has been given to DNA hydroxymethylation (5-hydroxymethylcytosine [5hmC]), the oxidized form of 5mC. Whereas 5mC is generally associated with the inhibition of gene expression, 5hmC has been associated with increased gene expression and is involved in cellular processes such as differentiation, development, and aging. Recent findings point toward a role for 5hmC in the development of diseases including AD, potentially opening new pathways for treating AD through correcting methylation and hydroxymethylation alterations. In the present study, levels of 5mC and 5hmC were investigated in the human middle frontal gyrus (MFG) and middle temporal gyrus (MTG) by immunohistochemistry. Immunoreactivity for 5mC and 5hmC were significantly increased in AD MFG (N = 13) and MTG (N = 29) compared with age-matched controls (MFG, N = 13 and MTG, N = 29). Global levels of 5mC and 5hmC positively correlated with each other and with markers of AD including amyloid beta, tau, and ubiquitin loads. Our results showed a global hypermethylation in the AD brain and revealed that levels of 5hmC were also significantly increased in AD MFG and MTG with no apparent influence of gender, age, postmortem delay, or tissue storage time. Using double-fluorescent immunolabeling, we found that in control and AD brains, levels of 5mC and 5hmC were low in astrocytes and microglia but were elevated in neurons. In addition, our colocalization study showed that within the same nuclei, 5mC and 5hmC mostly do not coexist. The present study clearly demonstrates the involvement of 5mC and 5hmC in AD emphasizing the need for future studies determining the exact time frame of these epigenetic changes during the progression of AD pathology.     

Oxidatively modified nucleic acids in preclinical Alzheimer's disease (PCAD) brain

Previous studies show increased oxidative DNA and RNA damage and diminished 8-oxoguanine glycosylase (OGG1) mediated base excision repair in vulnerable brain regions of mild cognitive impairment and late-stage Alzheimer's disease (LAD) subjects compared to normal control (NC) subjects. Recently, a preclinical stage of AD (PCAD) has been described in which subjects show no overt clinical manifestations of AD but demonstrate significant AD pathology at autopsy. To determine if DNA or RNA oxidation are significantly elevated in PCAD brain we quantified 8-hydroxyguanine (8-OHG) in sections of hippocampus/parahippocamapal gyri in PCAD and NC subjects using immunohistochemistry and confocal microscopy and in superior and middle temporal gyri (SMTG) using gas chromatography/mass spectrometry. To determine if increased DNA oxidation is associated with altered repair capacity, levels of OGG1 protein in HPG were measured by immunohistochemistry and levels of OGG1 mRNA were measured in SMTG using quantitative PCR. Results show significantly increased (p<0.05) 8-OHG immunostaining in DNA and RNA of PCAD HPG and significantly increased 8-OHG in PCAD SMTG. Quantification of OGG1 showed significantly elevated mRNA in PCAD SMTG and a trend toward elevated protein immunostaining in PCAD HPG. Overall, the data suggest oxidative damage to nucleic acids and a compensatory increase in OGG1 expression occur early in the pathogenesis of AD.     

Protein alterations in olfactory neuroblasts from Alzheimer donors

Definitive diagnosis of Alzheimer's disease (AD) is made by pathologic examination of postmortem brain tissue in conjunction with a clinical history of dementia. To date, there are no good biological markers for a positive diagnosis of AD in the living patient. In an effort to identify biological markers useful both in the clinical and pathologic diagnosis of AD, we have investigated disease-specific protein alterations in cultured olfactory neurons. Olfactory neurons are readily accessible by biopsy, can be propagated in primary cell culture as olfactory neuroblasts (ONs), and exhibit several elements of AD brain pathophysiology making them powerful tools for the study of AD. Two-dimensional gel analysis of ON proteins from neuropsychologically evaluated AD donors revealed a set of five proteins (Mr 17–50 kD, pI 4.8–6.7) that were significantly altered in concentration when compared to cells from age-matched controls. Further characterization and microsequence analysis could lead to the identification of proteins that may have important diagnostic or therapeutic value in the treatment of AD.     

Divergent behaviour of oxidative stress markers 8-hydroxydeoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (HNE) in breast carcinogenesis

Karihtala P, Kauppila S, Puistola U & Jukkola‐Vuorinen A
(2011) Histopathology 58, 854–862
Divergent behaviour of oxidative stress markers 8‐hydroxydeoxyguanosine (8‐OHdG) and 4‐hydroxy‐2‐nonenal (HNE) in breast carcinogenesis Aims: To clarify the role of oxidative stress during breast carcinogenesis by studying the expression of 8‐hydroxydeoxyguanosine (8‐OHdG) (a marker of oxidative DNA damage) and 4‐hydroxy‐2‐nonenal (HNE) (a marker of lipid peroxidation) during the different phases of breast carcinogenesis. Methods and results: The study material consisted of a total of 219 patients: 31 with usual ductal hyperplasia (UDH), 25 with atypical ductal hyperplasia (ADH), 30 with ductal carcinoma in situ (DCIS) and 133 with invasive carcinoma. The expression of 8‐OHdG and HNE were evaluated immunohistochemically. Both 8‐OHdG (77.4%) and HNE (45.8%) expression was already seen in UDH lesions. Interestingly, the trend of these two immunostainings during breast carcinogenesis was diverse. 8‐OHdG expression diminished significantly in invasive breast carcinomas compared to non‐invasive lesions (P < 0.005 when set against non‐invasive cohorts). Also within the same lesions, 8‐OHdG expression was the most intensive in benign cells. Conversely, HNE immunostaining was strongest in invasive breast carcinomas (UDH versus invasive cohort, P = 0.015). Conclusions: 4‐hydroxy‐2‐nonenal as a marker of lipid peroxidation increases during breast carcinogenesis, reflecting the role of oxidative stress in the pathogenesis of breast cancer. However, 8‐OHdG shows diminished levels in carcinomas, possibly resulting from the induction of DNA repair in these invasive lesions.