An Assessment of Oxidative Damage to Proteins, Lipids, and DNA in Brain from Patients with Alzheimer's Disease

Abstract: Oxidative stress may contribute to neuronal loss in Alzheimer's disease (AD). The present study compares the levels of oxidative damage to proteins, lipids, and DNA bases from seven different brain areas of AD and matched control tissues by using a range of techniques. No differences in levels of lipid peroxidation were found in any of the brain regions by using two different assay systems. Overall, there was a trend for protein carbonyl levels to be increased in AD in frontal, occipital, parietal, and temporal lobe, middle temporal gyrus, and hippocampus, but a significant difference was found only in the parietal lobe. Gas chromatography-mass spectrometry was used to measure products of damage to all four DNA bases. Increased levels of some (8-hydroxyadenine, 8-hydroxyguanine, thymine glycol, Fapy-guanine, 5-hydroxyuracil, and Fapy-adenine), but not all, oxidized DNA bases were observed in parietal, temporal, occipital, and frontal lobe, superior temporal gyrus, and hippocampus. The baseline level of oxidative DNA damage in the temporal lobe was higher than in other brain regions in both control and AD brain. The finding of increased oxidative damage to protein and DNA strengthens the possibility that oxidative damage may play a role in the pathogenesis of AD in at least some key brain regions.     

Increased oxidative damage in nuclear and mitochondrial DNA in Alzheimer's disease

Increasing evidence suggests that oxidative stress is associated with normal aging and several neurodegenerative diseases, including Alzheimer's disease (AD). Here we quantified multiple oxidized bases in nuclear and mitochondrial DNA of frontal, parietal, and temporal lobes and cerebellum from short postmortem interval AD brain and age-matched control subjects using gas chromatography/mass spectrometry with selective ion monitoring (GC/MS-SIM) and stable labeled internal standards. Nuclear and mitochondrial DNA were extracted from eight AD and eight age-matched control subjects. We found that levels of multiple oxidized bases in AD brain specimens were significantly (p < 0.05) higher in frontal, parietal, and temporal lobes compared to control subjects and that mitochondrial DNA had approximately 10-fold higher levels of oxidized bases than nuclear DNA. These data are consistent with higher levels of oxidative stress in mitochondria. Eight-hydroxyguanine, a widely studied biomarker of DNA damage, was approximately 10-fold higher than other oxidized base adducts in both AD and control subjects. DNA from temporal lobe showed the most oxidative damage, whereas cerebellum was only slightly affected in AD brains. These results suggest that oxidative damage to mitochondrial DNA may contribute to the neurodegeneration of AD.     

Increased Nuclear DNA Oxidation in the Brain in Alzheimer's Disease

Abstract: Multiple lines of evidence indicate that oxidative stress is a contributor to neuronal death in Alzheimer's disease (AD). The oxidative damage that occurs to DNA may play a role in both normal aging and neurodegenerative diseases, including AD. This is a study of the oxidative damage that occurs in nuclear DNA in the brains of AD patients and cognitively intact, prospectively evaluated, age-matched control subjects. Nuclear DNA from frontal, temporal, and parietal lobes and cerebellum was isolated from 11 control subjects and 9 AD subjects, and oxidized purine and pyrimidine bases were quantitated using gas chromatography/mass spectrometry. Stable isotope-labeled oxidized base analogues were used as internal standards to measure 5-hydroxyuracil, 5-hydroxycytosine, 8-hydroxyadenine, 4,6-diamino-5-formamidopyrimidine (Fapy-adenine), 8-hydroxyguanine, and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-guanine). Statistically significant elevations of 5-hydroxycytosine, 5-hydroxyuracil, 8-hydroxyadenine, and 8-hydroxyguanine were found in AD brain compared with control subjects ( p < 0.05). There was an increased trend in the levels of Fapy-adenine in the AD brain, and Fapy-guanine showed a trend toward higher levels in control brains compared with AD. A generally higher level of oxidative DNA damage was present in neocortical regions than cerebellum. No significant correlation was observed between the oxidized bases and neurofibrillary tangle and senile plaque counts. Our results demonstrate that nuclear DNA damage by oxygen-derived radicals is increased in AD and support the concept that the brain is under increased oxidative stress in AD.     

Increased oxidative damage in nuclear and mitochondrial DNA in mild cognitive impairment

Increasing evidence suggests that oxidative damage is associated with normal aging and several neurodegenerative diseases. Mild cognitive impairment (MCI), the phase between normal aging and early dementia, is a common problem in the elderly with many subjects going on to develop Alzheimer's disease (AD). Although increased DNA oxidation is observed in the AD brain, it is unclear when the oxidative damage begins. To determine if DNA oxidation occurs in the brain of subjects with MCI, we quantified multiple oxidized bases in nuclear and mitochondrial DNA isolated from frontal, parietal and temporal lobes and cerebellum of short post-mortem interval autopsies of eight amnestic patients with MCI and six age-matched control subjects using gas chromatography/mass spectrometry with selective ion monitoring. We found statistically significant elevations (p < 0.05) of 8-hydroxyguanine, a widely studied biomarker of DNA damage, in MCI nuclear DNA from frontal and temporal lobe and in mitochondrial DNA from the temporal lobe compared with age-matched control subjects. Levels of 8-hydroxyadenine and 4,6-diamino-5-formamidopyrimidine were significantly elevated in nuclear DNA from all three neocortical regions in MCI. Statistically significant elevations of 4,6-diamino-5-formamidopyrimidine were also observed in mitochondrial DNA of MCI temporal, frontal and parietal lobes. These results suggest that oxidative damage to nuclear and mitochondrial DNA occurs in the earliest detectable phase of AD and may play a meaningful role in the pathogenesis of this disease.     

Isofurans,but not F2-isoprostanes,are increased in the substantia nigra of patients with Parkinson's disease and with dementia with Lewy body disease

F2-isoprostanes (F2-IsoPs) are well-established sensitive and specific markers of oxidative stress in vivo. Isofurans (IsoFs) are also products of lipid peroxidation, but in contrast to F2-IsoPs, their formation is favored when oxygen tension is increased in vitro or in vivo. Mitochondrial dysfunction in Parkinson's disease (PD) may not only lead to oxidative damage to brain tissue but also potentially result in increased intracellular oxygen tension, thereby influencing relative concentrations of F2-IsoPs and IsoFs. In this study, we attempted to compare the levels of F2-IsoPs and IsoFs esterified in phospholipids in the substantia nigra (SN) from patients with PD to those of age-matched controls as well as patients with other neurodegenerative diseases, including dementia with Lewy body disease (DLB), multiple system atrophy (MSA), and Alzheimer's disease (AD). The results demonstrated that IsoFs but not F2-IsoPs in the SN of patients with PD and DLB were significantly higher than those of controls. Levels of IsoFs and F2-IsoPs in the SN of patients with MSA and AD were indistinguishable from those of age-matched controls. This preferential increase in IsoFs in the SN of patients with PD or DLB not only indicates a unique mode of oxidant injury in these two diseases but also suggests different underlying mechanisms of dopaminergic neurodegeneration in PD and DLB from those of MSA.     

Structural Alteration of the Dorsal Visual Network in DLB Patients with Visual Hallucinations: A Cortical Thickness MRI Study

Visual hallucinations (VH) represent one of the core features in discriminating dementia with Lewy bodies (DLB) from Alzheimer’s Disease (AD). Previous studies reported that in DLB patients functional alterations of the parieto-occipital regions were correlated with the presence of VH. The aim of our study was to assess whether morphological changes in specific cortical regions of DLB could be related to the presence and severity of VH. We performed a cortical thickness analysis on magnetic resonance imaging data in a cohort including 18 DLB patients, 15 AD patients and 14 healthy control subjects. Relatively to DLB group, correlation analysis between the cortical thickness and the Neuropsychiatric Inventory (NPI) hallucination item scores was also performed. Cortical thickness was reduced bilaterally in DLB compared to controls in the pericalcarine and lingual gyri, cuneus, precuneus, superior parietal gyrus. Cortical thinning was found bilaterally in AD compared to controls in temporal cortex including the superior and middle temporal gyrus, part of inferior temporal cortex, temporal pole and insula. Inferior parietal and supramarginal gyri were also affected bilaterally in AD as compared to controls. The comparison between DLB and AD evidenced cortical thinning in DLB group in the right posterior regions including superior parietal gyrus, precuneus, cuneus, pericalcarine and lingual gyri. Furthermore, the correlation analysis between cortical thickness and NPI hallucination item scores showed that the structural alteration in the dorsal visual regions including superior parietal gyrus and precuneus closely correlated with the occurrence and severity of VH. We suggest that structural changes in key regions of the dorsal visual network may play a crucial role in the physiopathology of VH in DLB patients.     

Evidence of Increased Oxidative Damage in Both Sporadic and Familial Amyotrophic Lateral Sclerosis

Abstract: Some cases of autosomal dominant familial amyotrophic lateral sclerosis (FALS) are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1), suggesting that oxidative damage may play a role in ALS pathogenesis. To further investigate the biochemical features of FALS and sporadic ALS (SALS), we examined markers of oxidative damage to protein, lipids, and DNA in motor cortex (Brodmann area 4), parietal cortex (Brodmann area 40), and cerebellum from control subjects, FALS patients with and without known SOD mutations, SALS patients, and disease controls (Pick's disease, progressive supranuclear palsy, diffuse Lewy body disease). Protein carbonyl and nuclear DNA 8-hydroxy-2'-deoxyguanosine (OH⁸dG) levels were increased in SALS motor cortex but not in FALS patients. Malondialdehyde levels showed no significant changes. Immunohistochemical studies showed increased neuronal staining for hemeoxygenase-1, malondialdehyde-modified protein, and OH⁸dG in both SALS and FALS spinal cord. These studies therefore provide further evidence that oxidative damage may play a role in the pathogenesis of neuronal degeneration in both SALS and FALS.     

Formation of the base modification 8-hydroxyl-2'-deoxyguanosine and DNA fragmentation following seizures induced by systemic kainic acid in the rat

The formation of oxidative DNA damage as a consequence of seizures remains little explored. We therefore investigated the regional and temporal profile of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) formation, a hallmark of oxidative DNA damage and DNA fragmentation in rat brain following seizures induced by systemic kainic acid (KA). Formation of 8-OHdG was determined via HPLC with electrochemical detection, and single- and double-stranded DNA breaks were detected using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated nick end-labeling (TUNEL), respectively. Systemic KA (11 mg/kg) significantly increased levels of 8-OHdG within the thalamus after 2 h, within the amygdala/piriform cortex after 4 h, and within the hippocampus after 8 h. Levels remained elevated up to sevenfold within these areas for 72 h. Smaller increases in 8-OHdG levels were also detected within the parietal cortex and striatum. PANT-positive cells were detected within the thalamus, amygdala/piriform cortex, and hippocampus 24-72 h following KA injection. TUNEL-positive cells appeared within the same brain regions and over a similar time course (24-72 h) but were generally lower in number. The present data suggest oxidative damage to DNA may be an early consequence of epileptic seizures and a possible initiation event in the progression of seizure-induced injury to DNA fragmentation and cell death.     

The steady-state levels of oxidative DNA damage and of lipid peroxidation (F2-isoprostanes) are not correlated in healthy human subjects

Oxidative damage to DNA in human tissues can be determined by measuring multiple products of oxidative damage to the purine and pyrimidine bases using gas chromatography-mass spectrometry (GC-MS). Oxidative damage to lipids (lipid peroxidation) can be quantitated by the mass spectrometry-based determination of F2-isoprostanes, specific end-products of the peroxidation of arachidonic acid residues in lipids. For both DNA base damage products and 8-epi prostaglandin F2alpha (PGF2alpha), there is a wide variation in levels between different healthy human subjects. We measured multiple products of oxidative damage to DNA bases in white cells, and 8-epi PGF2alpha in plasma, from blood samples obtained from healthy human subjects in the UK and in Portugal. No correlation of 8-epi PGF2alpha levels with levels of any modified DNA base (including 8-hydroxyguanine) was observed. We conclude that no single parameter can be measured as an index of "oxidative stress" or "oxidative damage" in vivo.     

Oxidative DNA damage in polymorphonuclear leukocytes of patients with familial Mediterranean fever

Familial Mediterranean fever (FMF) is an autosomal recessively inherited disorder characterized by recurrent, inflammatory self-limited episodes of fever and other symptoms. This disease is caused by more than 25 mutations in the gene MEFV. During fever attacks, there is a substantial influx of polymorphonuclear leukocytes into the affected tissues. Attack-free periods are accompanied by the up-regulation of neutrophil and monocyte phagocytic activity and oxidative burst. These facts led us to hypothesize that oxidative damage by free radicals to DNA may accumulate in FMF patients. To test this hypothesis, we investigated oxidative DNA damage in polymorphonuclear leukocytes of FMF patients during the attack-free period in comparison with FMF-free control individuals. DNA was isolated from polymorphonuclear leukocytes of 17 FMF patients and 10 control individuals. DNA samples were analyzed by liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure the levels of various typical oxidatively induced products of DNA. We show, for the first time, that FMF patients accumulate statistically significant levels of these lesions in their DNA when compared to FMF-free control individuals. This work suggests that the persistent oxidative stress with excess production of free radicals in FMF patients may lead to accumulation of oxidative DNA damage. Defective DNA repair may also contribute to this phenomenon, perhaps due to mutations in the MEFV gene. The accumulation of mutagenic and cytotoxic DNA lesions may contribute to increased mutations and apoptosis in FMF patients, thus to worsening of the disease and well-being of the patients. Future research should deal with prevention of oxidative DNA damage and apoptosis in FMF patients, and also the elucidation of a possible role of DNA repair in this disease.     

Room temperature derivatization of 5-hydroxy-2'- deoxycytidine and 5-hydroxymethyl-2'-deoxyuridine for analysis by GC/MS

Hydroxylated DNA basesare one type of oxygen free radical-induced damage to DNA. Such damage has been implicated in the process of carcinogenesis, and the levels of hydroxylated DNA bases may serve as a marker of cancer risk in humans. Measurement of oxidative DNA damage can be hampered by the ease with which artifactual oxidative DNA damage can be induced via sample processing. In this report we describe convenient room temperature derivatization and stability of 5-hydroxy-2'-deoxycytidine (5-OHdCyd) and 5-hydroxymethyl-2'-deoxyuridine (5-OHmdU) using GC/MS analysis. The derivatization reagent was N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1% trimethylchloro-silane:acetonitrile, 2:1. This method avoids use of acid and is much milder than previously reported derivatization conditions which typically involve heating above 100C for at least 20 min. Although heating has been reported to be problematic, the calculated levels of 5-OHdCyd and 5-OHmdU in enzymatically-hydrolysed calf thymus DNA were very similar in our hands with and without heating the sample for 20 min. As an example of the technique, comparison of 5-OHdCyd and 5-OHmdU levels in calf thymus DNA indicated relatively higher endogenous levels of 5-OHdCyd. In DNA treated with hydrogen peroxide and ferric chloride, however, the levels of 5-OHmdU increased much more than that of 5-OHdCyd. In addition to these hydroxylated derivatives of deoxycytidine and thymidine, the method also appears to work well with 8-oxoguanine, 4,6-diamino-5-(formylamino)pyrimidine, and 5-methyl-2'-deoxycytidine. This method may therefore be useful with a variety of modified DNA bases and nucleosides.     

DNA damage and repair in type 2 diabetes mellitus

DNA damage may be associated with type 2 diabetes mellitus (T2DM) and its complications mainly through oxidative stress. Little is known about DNA repair disturbances potentially contributing to the overall extent of DNA damage in T2DM, which, in turn, may be linked with genomic instability resulting in cancer. To assess whether DNA repair may be perturbed in 2DM we determined: (1) the level of endogenous basal DNA damage, this means damage recognized in the alkaline comet assay (DNA strand breaks and alkali labile sites) as well as endogenous oxidative and alkylative DNA damage (2) the sensitivity to DNA-damaging agents hydrogen peroxide and doxorubicin and the efficacy of removing of DNA damage induced by these agents in peripheral blood lymphocytes of T2DM patients and healthy individuals. The level of DNA damage and the kinetics of DNA repair was evaluated by the alkaline single cell gel electrophoresis (comet assay). Oxidative and alkylative DNA damage were assayed with the use of DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. The levels of basal endogenous and oxidative DNA damage in diabetes patients were higher than in control subjects. There was no difference between the level of alkylative DNA in the patients and the controls. Diabetes patients displayed higher susceptibility to hydrogen peroxide and doxorubicin and decreased efficacy of repairing DNA damage induced by these agents than healthy controls. Our results suggest that type 2 diabetes mellitus may be associated not only with the elevated level of oxidative DNA damage but also with the increased susceptibility to mutagens and the decreased efficacy of DNA repair. These features may contribute to a link between diabetes and cancer and metrics of DNA damage and repair, measured by the comet assay, may be markers of risk of cancer in diabetes.     

Neurodegeneration-associated instability of ribosomal DNA

Homologous recombination (HR)-mediated instability of the repetitively organized ribosomal DNA (rDNA) has been proposed as a mediator of cell senescence in yeast triggering the DNA damage response. High individual variability in the content of human rDNA suggests that this genomic region remained relatively unstable throughout evolution. Therefore, quantitative real-time polymerase chain reaction was used to determine the genomic content of rDNA in post mortem samples of parietal cortex from 14 young and 9 elderly individuals with no diagnosis of a chronic neurodegenerative/neurological disease. In addition, rDNA content in that brain region was compared between 10 age-matched control individuals and 10 patients with dementia with Lewy bodies (DLB) which involves neurodegeneration of the cerebral cortex. Probing rRNA-coding regions of rDNA revealed no effects of aging on the rDNA content. Elevated rDNA content was observed in DLB. Conversely, in the DLB pathology-free cerebellum, lower genomic content of rDNA was present in the DLB group. In the parietal cortex, such a DLB-associated instability of rDNA was not accompanied by any major changes of cytosine–phosphate–guanine methylation of the rDNA promoter. As increased cerebro-cortical rDNA content was previously reported in Alzheimer's disease, neurodegeneration appears to be associated with instability of rDNA. The hypothetical origins and consequences of this phenomenon are discussed including possibilities that the DNA damage-induced recombination destabilizes rDNA and that differential content of rDNA affects heterochromatin formation, gene expression and/or DNA damage response. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.     

The steady-state levels of oxidative DNA damage and of lipid peroxidation (F2-isoprostanes) are not correlated in healthy human subjects

Oxidative damage to DNA in human tissues can be determined by measuring multiple products of oxidative damage to the purine and pyrimidine bases using gas chromatography-mass spectrometry (GC-MS). Oxidative damage to lipids (lipid peroxidation) can be quantitated by the mass spectrometry-based determination of F₂-isoprostanes, specific end-products of the peroxidation of arachidonic acid residues in lipids. For both DNA base damage products and 8-epi prostaglandin F_2α (PGF_2α), there is a wide variation in levels between different healthy human subjects. We measured multiple products of oxidative damage to DNA bases in white cells, and 8-epi PGF_2α in plasma, from blood samples obtained from healthy human subjects in the UK and in Portugal. No correlation of 8-epi PGF_2α levels with levels of any modified DNA base (including 8-hydroxyguanine) was observed. We conclude that no single parameter can be measured as an index of “oxidative stress” or “oxidative damage” in vivo.     

The mechanisms of oxidative DNA damage and apoptosis induced by norsalsolinol, an endogenous tetrahydroisoquinoline derivative associated with Parkinson's disease

Tetrahydroisoquinoline (TIQ) derivatives are putative neurotoxins that may contribute to the degeneration of dopaminergic neurons in Parkinson's disease. One TIQ, norsalsolinol (NorSAL), is present in dopamine-rich areas of human brain, including the substantia nigra. Here, we demonstrate that NorSAL reduces cell viability and induces apoptosis via cytochrome c release and caspase 3 activation in SH-SY5Y human neuroblastoma cells. Cytochrome c release, caspase 3 activation, and apoptosis induction were all inhibited by the antioxidant N -acetylcysteine. Thus, reactive oxygen species (ROS) contribute to apoptosis induced by NorSAL. Treatment with NorSAL also increased levels of oxidative damage to DNA, a stimulus for apoptosis, in SH-SY5Y. To clarify the mechanism of intracellular DNA damage, we examined the DNA damage caused by NorSAL using ³²P-5'-end-labeled isolated DNA fragments. NorSAL induced DNA damage in the presence of Cu(II). Catalase and bathocuproine, a Cu(I) chelator, inhibited this DNA damage, suggesting that ROS such as the Cu(I)-hydroperoxo complex derived from the reaction of H₂O₂ with Cu(I), promote DNA damage by NorSAL. In summary, NorSAL-generated ROS induced oxidative DNA damage, which led to caspase-dependent apoptosis in neuronal cells.     

Human brain cortex: mitochondrial oxidative damage and adaptive response in Parkinson disease and in dementia with Lewy bodies

Frontal cortex samples from frozen human brains were used to assess tissue respiration; content of mitochondria; mitochondrial oxygen uptake; activity of respiratory complexes and of mitochondrial nitric oxide synthase (mtNOS); content of cytochromes a, b, and c; oxidative damage (protein carbonyls and TBARS); and expression of Mn-SOD in patients with Parkinson disease (PD) and with dementia with Lewy bodies (DLB) in comparison with those of normal healthy controls. Brain cortex and mitochondrial O₂ uptake and complex I activity were significantly lower in PD and DLB, whereas mtNOS activity, cytochrome content, expression of Mn-SOD, mitochondrial mass, and oxidative damage were significantly higher in the frontal cortex in PD and DLB. The decreases in tissue and mitochondrial O₂ uptake and in complex I activity are considered the consequences of mitochondrial oxidative damage. The increases in mtNOS activity and in mitochondrial mass are interpreted as an adaptive response of the frontal cortex that involves increased NO signaling for mitochondrial biogenesis. The adaptive response would partially compensate for mitochondrial dysfunction in these neurodegenerative diseases and would afford a human evolutionary response to shortage of ATP in the frontal cortex.     

Levels of oxidative damage and proinflammatory cytokines are enhanced in patients with active vitiligo

Vitiligo is an autoimmune depigmenting skin disease characterised by loss of melanocytes wherein oxidative stress is proposed to be the initial triggering factor with subsequent immune dysregulation. This study aimed to evaluate the relationship, if any, between the generation of reactive oxygen species (ROS), markers of oxidative damage and circulating cytokines in patients with active vitiligo. The generation of ROS in erythrocytes and neutrophils was significantly higher in patients with active vitiligo than healthy controls. Alongside, markers of oxidative stress-mediated damage namely lipid peroxidation, DNA damage and protein carbonylation were evaluated. Patients with active vitiligo demonstrated increased lipid and DNA damage but minimal protein damage. There was a significant decline in the free radical scavenging capacity of active vitiligo cases. A positive correlation existed between baseline levels of ROS and lipid peroxidation as also DNA damage. Patients with active vitiligo demonstrated an increase in several proinflammatory (IL-6, TNF-α, IL-1β, IFN-γ and IL-8) and some anti-inflammatory/immunoregulatory (IL-5 and IL-10) cytokines. Importantly, the levels of IFN-γ and IL-10 consistently correlated with the generation of ROS, markers of damage and their free radical scavenging capacity. Taken together, patients with active vitiligo demonstrated an enhanced generation of ROS in erythrocytes and neutrophils which mediated lipid peroxidation, DNA damage and coupled with a decline in their antioxidant capacity created a pro-oxidant milieu that favoured tissue damage and potential generation of neoantigens, accounting for disease progression.     

Oxidative damage to DNA and single strand break repair capacity: relationship to other measures of oxidative stress in a population cohort

It is well accepted that oxidative DNA repair capacity, oxidative damage to DNA and oxidative stress play central roles in aging and disease development. However, the correlation between oxidative damage to DNA, markers of oxidant stress and DNA repair capacity is unclear. In addition, there is no universally accepted panel of markers to assess oxidative stress in humans. Our interest is oxidative damage to DNA and its correlation with DNA repair capacity and other markers of oxidative stress. We present preliminary data from a small comet study that attempts to correlate single strand break (SSB) level with single strand break repair capacity (SSB-RC) and markers of oxidant stress and inflammation. In this limited study of four very small age-matched 24-individual groups of male and female whites and African-Americans aged 30-64 years, we found that females have higher single strand break (SSB) levels than males (p=0.013). There was a significant negative correlation between SSB-RC and SSB level (p=0.041). There was a positive correlation between SSBs in African American males with both heme degradation products (p=0.008) and high-sensitivity C-reactive protein (hs-CRP) (p=0.022). We found a significant interaction between hs-CRP and sex in their effect on residual DNA damage (p=0.002). Red blood cell reduced glutathione concentration was positively correlated with the levels of oxidized bases detected by endonuclease III (p=0.047), heme degradation products (p=0.015) and hs-CRP (p=0.020). However, plasma carbonyl levels showed no significant correlation with other markers. The data from the literature and from our very limited study suggest a complex relationship between measures of oxidative stress and frequently used clinical parameters believed to reflect inflammation or oxidative stress.     

Endogenous oxidative DNA base modifications analysed with repair enzymes and GC/MS technique

GC/MS technique was used to identify endogenous levels of oxidatively modified DNA bases. To avoid possible artefact formation we used Fpg and Endo III endonucleases instead of acid hydrolysis to liberate the base products from unmodified DNA samples. Several different DNA preparations were used: (i) commercial calf thymus DNA, (ii) DNA isolated from rat liver, (iii) DNA isolated from human lymphocytes and (iv) nuclei isolated from rat liver. In all DNA samples used in our assays the most efficiently removed bases by Fpg protein are FapyG and FapyA although 8-oxoG was also detected in all preparations. The amount of 8-oxoG in human lymphocytes and in rat liver DNA was 3 and 2 per 10(7)bases, respectively. It is reasonable to postulate that the presented method is one of the techniques which should be used to reveal the enigma of endogenous, oxidative DNA damage.