Immunochemical detection of oxidatively damaged DNA

Abstract

Oxidatively damaged DNA is implicated in various diseases, including neurodegenerative disorders, cancer, diabetes, cardiovascular and inflammatory diseases as well as aging. Several methods have been developed to detect oxidatively damaged DNA. They include chromatographic techniques, the Comet assay, ³²P-postlabelling and immunochemical methods that use antibodies to detect oxidized lesions. In this review, we discuss the detection of 8-oxo-7,8-dihydro-29-deoxyguanosine (8-oxodG), the most abundant oxidized nucleoside. This lesion is frequently used as a marker of exposure to oxidants, including environmental pollutants, as well as a potential marker of disease progression. We concentrate on studies published between the years 2000 and 2011 that used enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry to detect 8-oxodG in humans, laboratory animals and in cell lines. Oxidative damage observed in these organisms resulted from disease, exposure to environmental pollutants or from in vitro treatment with various chemical and physical factors.     

Immunochemical detection of oxidatively damaged DNA

Oxidatively damaged DNA is implicated in various diseases, including neurodegenerative disorders, cancer, diabetes, cardiovascular and inflammatory diseases as well as aging. Several methods have been developed to detect oxidatively damaged DNA. They include chromatographic techniques, the Comet assay, (32)P-postlabelling and immunochemical methods that use antibodies to detect oxidized lesions. In this review, we discuss the detection of 8-oxo-7,8-dihydro-29-deoxyguanosine (8-oxodG), the most abundant oxidized nucleoside. This lesion is frequently used as a marker of exposure to oxidants, including environmental pollutants, as well as a potential marker of disease progression. We concentrate on studies published between the years 2000 and 2011 that used enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry to detect 8-oxodG in humans, laboratory animals and in cell lines. Oxidative damage observed in these organisms resulted from disease, exposure to environmental pollutants or from in vitro treatment with various chemical and physical factors.     

Processing oxidatively damaged bases at DNA strand breaks by APE1

Reactive oxygen species attack the structure of DNA, thus altering its base-pairing properties. Consequently, oxidative stress-associated DNA lesions are a major source of the mutation load that gives rise to cancer and other diseases. Base excision repair (BER) is the pathway primarily tasked with repairing DNA base damage, with apurinic/apyrimidinic endonuclease (APE1) having both AP-endonuclease and 3′ to 5′ exonuclease (exo) DNA cleavage functions. The lesion 8-oxo-7,8-dihydroguanine (8-oxoG) can enter the genome as either a product of direct damage to the DNA, or through polymerase insertion at the 3′-end of a DNA strand during replication or repair. Importantly, 3′-8-oxoG impairs the ligation step of BER and therefore must be removed by the exo activity of a surrogate enzyme to prevent double stranded breaks and cell death. In the present study, we use X-ray crystallography to characterize the exo activity of APE1 on 3′-8-oxoG substrates. These structures support a unified APE1 exo mechanism that differs from its more canonical AP-endonuclease activity. In addition, through complementation of the structural data with enzyme kinetics and binding studies employing both wild-type and rationally designed APE1 mutants, we were able to identify and characterize unique protein: DNA contacts that specifically mediate 8-oxoG removal by APE1.     

Stereoselective excision of thymine glycol from oxidatively damaged DNA

DNA damage created by reactive oxygen species includes the prototypic oxidized pyrimidine, thymine glycol (Tg), which exists in oxidatively damaged DNA as two diastereoisomeric pairs. In Escherichia coli, Saccharomyces cerevesiae and mice, Tg is preferentially excised by endonuclease III (Endo III) and endonuclease VIII (Endo VIII), yNTG1 and yNTG2, and mNTH and mNEIL1, respectively. We have explored the ability of these DNA glycosylases to discriminate between Tg stereoisomers. Oligonucleotides containing a single, chromatographically pure (5S,6R) or (5R,6S) stereoisomer of Tg were prepared by oxidation with osmium tetroxide. Steady-state kinetic analyses of the excision process revealed that Endo III, Endo VIII, yNTG1, mNTH and mNEIL1, but not yNTG2, excise Tg isomers from DNA in a stereoselective manner, as reflected in the parameter of catalytic efficiency (k_cat/K_m). When DNA glycosylases occur as complementary pairs, failure of one or both enzymes to excise their cognate Tg stereoisomer from oxidatively damaged DNA could have deleterious consequences for the cell.     

Assays for urinary biomarkers of oxidatively damaged nucleic acids

Abstract

The analysis of oxidized nucleic acid metabolites can be performed by a variety of methodologies: liquid chromatography coupled with electrochemical or mass-spectrometry detection, gas chromatography coupled with mass spectrometry, capillary electrophoresis and ELISA (Enzyme-linked immunosorbent assay). The major analytical challenge is specificity. The best combination of selectivity and speed of analysis can be obtained by liquid chromatography coupled with tandem mass spectrometric detection. This, however, is also the most demanding technique with regard to price, complexity and skills requirement. The available ELISA methods present considerable specificity problems and cannot be recommended at present. The oxidized nucleic acid metabolites in urine are assumed to originate from the DNA and RNA. However, direct evidence is not available. A possible contribution from the nucleotide pools is most probably minimal, if existing. Recent investigation on RNA oxidation has shown conditions where RNA oxidation but not DNA oxidation is prominent, and while investigation on DNA is of huge interest, RNA oxidation may be overlooked. The methods for analyzing oxidized deoxynucleosides can easily be expanded to analyze the oxidized ribonucleosides. The urinary measurement of oxidized nucleic acid metabolites provides a non-invasive measurement of oxidative stress to DNA and RNA.     

Assays for urinary biomarkers of oxidatively damaged nucleic acids

The analysis of oxidized nucleic acid metabolites can be performed by a variety of methodologies: liquid chromatography coupled with electrochemical or mass-spectrometry detection, gas chromatography coupled with mass spectrometry, capillary electrophoresis and ELISA (Enzyme-linked immunosorbent assay). The major analytical challenge is specificity. The best combination of selectivity and speed of analysis can be obtained by liquid chromatography coupled with tandem mass spectrometric detection. This, however, is also the most demanding technique with regard to price, complexity and skills requirement. The available ELISA methods present considerable specificity problems and cannot be recommended at present. The oxidized nucleic acid metabolites in urine are assumed to originate from the DNA and RNA. However, direct evidence is not available. A possible contribution from the nucleotide pools is most probably minimal, if existing. Recent investigation on RNA oxidation has shown conditions where RNA oxidation but not DNA oxidation is prominent, and while investigation on DNA is of huge interest, RNA oxidation may be overlooked. The methods for analyzing oxidized deoxynucleosides can easily be expanded to analyze the oxidized ribonucleosides. The urinary measurement of oxidized nucleic acid metabolites provides a non-invasive measurement of oxidative stress to DNA and RNA.     

Aging and oxidatively damaged nuclear DNA in animal organs

Oxidative stress is considered to contribute to aging and is associated with the generation of oxidatively damaged DNA, including 8-oxo-7,8-dihydroguanine. We have identified 69 studies that have measured the level of oxidatively damaged DNA in organs of animals at various ages. In general, organs with limited cell proliferation, i.e., liver, kidney, brain, heart, pancreas, and muscle, tended to show accumulation of DNA damage with age, whereas organs with highly proliferating cells, such as intestine, spleen, and testis, showed more equivocal or no effect of age. A restricted analysis of studies reporting a baseline level of damaged DNA that was fewer than 5 lesions/10⁶ dG showed that 21 of 29 studies reported age-associated accumulation of DNA damage. The standardized mean difference in oxidatively damaged DNA between the oldest and the youngest age groups was 1.49 (95% CI 1.03–1.95). There was no difference between age span, number of tested organs, statistical power, sex, strain, or breeding between the studies showing positive and null effects. Citation and publication bias seems to be a problem in the overall dataset, whereas it is less pronounced in the restricted dataset. There is compelling evidence for aging-associated accumulation of oxidatively damaged DNA in organs with limited cell proliferation.     

Dietary antioxidants and beneficial effect on oxidatively damaged DNA

Many biomonitoring studies have investigated the role of antioxidants in reducing oxidatively generated DNA damage in urine and white blood cells. A collective interpretation is difficult because many studies lack sufficient control and have unreasonably high baseline levels of oxidatively damaged DNA. In a survey of this antioxidant hypothesis, we identified 139 cross-sectional and intervention studies. Restricted selection criteria with exclusion of studies having suboptimal design or unreasonably high baseline damage level provided 85 eligible studies for analysis. Ten of the 27 cross-sectional studies reported negative correlations between antioxidants and oxidatively damaged DNA, albeit with correlation coefficients explaining less than 20% of the variance. Sixty-two intervention studies reported mixed results, which did not depend on sample size or duration of the intervention. Reduced levels of oxidatively damaged DNA in white blood cells and urine were reported in far more studies than expected by chance alone. Supplementation with antioxidant-rich foods was more effective than that with single antioxidants in lowering urinary excretion of oxidatively damage DNA. In conclusion, this survey indicates that ingestion of antioxidants may be associated with reduced level of DNA damage in white blood cells and urine of humans, albeit the effect is lower than previously expected.     

The nuclear proteasome and the degradation of oxidatively damaged proteins

Summary. The accumulation of oxidized proteins is known to be linked to some severe neurodegenerative diseases like Alzheimer’s, Parkinson’s and Huntington’s disease. Furthermore, the aging process is also accompanied by an ongoing aggregation of misfolded and damaged proteins. Therefore, mammalian cells have developed potent degradation systems, which selectively degrade damaged and misfolded proteins. The proteasomal system is largely responsible for the removal of oxidatively damaged proteins form the cellular environment. Not only cytosolic proteins are prone to oxidative stress, also nuclear proteins are readily oxidized. The nuclear proteasomal system is responsible for the degradation of these proteins. This review is focused on the specific degradation of oxidized nuclear proteins, the role of the proteasome in this process and the regulation of the nuclear proteasomal system under oxidative conditions.     

Chemical and biological consequences of oxidatively damaged guanine in DNA

Of the four native nucleosides, 2'-deoxyguanosine (dGuo) is most easily oxidized. Two lesions derived from dGuo are 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy)?dGuo. Furthermore, while steady-state levels of 8-oxodGuo can be detected in genomic DNA, it is also known that 8-oxodGuo is more easily oxidized than dGuo. Thus, 8-oxodGuo is susceptible to further oxidation to form several hyperoxidized dGuo products. This review addresses the structural impact, the mutagenic and genotoxic potential, and biological implications of oxidatively damaged DNA, in particular 8-oxodGuo, Fapy?dGuo, and the hyperoxidized dGuo products.     

Chemical and biological consequences of oxidatively damaged guanine in DNA

Abstract

Of the four native nucleosides, 2′-deoxyguanosine (dGuo) is most easily oxidized. Two lesions derived from dGuo are 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy)?dGuo. Furthermore, while steady-state levels of 8-oxodGuo can be detected in genomic DNA, it is also known that 8-oxodGuo is more easily oxidized than dGuo. Thus, 8-oxodGuo is susceptible to further oxidation to form several hyperoxidized dGuo products. This review addresses the structural impact, the mutagenic and genotoxic potential, and biological implications of oxidatively damaged DNA, in particular 8-oxodGuo, Fapy?dGuo, and the hyperoxidized dGuo products.     

Accelerated accumulation of amyloid beta proteins on oxidatively damaged lipid membranes

The fully developed lesion of Alzheimer's Disease is a dense plaque composed of fibrillar amyloid beta-proteins with a characteristic and well-ordered beta-sheet secondary structure. Because the incipient lesion most likely develops when these proteins are first induced to form beta-sheet secondary structure, it is important to understand factors that induce amyloid beta-proteins to adopt this conformation. In this investigation we used a novel form of infrared spectroscopy that can characterize the conformation, orientation, and rate of accumulation of the protein on various lipid membranes to determine whether oxidatively damaged phospholipid membranes induce the formation of beta-sheet secondary structure in a 42-residue amyloid beta-protein. We found that membranes containing oxidatively damaged phospholipids accumulated amyloid beta-protein significantly faster than membranes containing only unoxidized or saturated phospholipids. Accelerated accumulation was also seen when 3 mol % G(M1) ganglioside was incorporated into a saturated phosphatidylcholine membrane. The accumulated protein more completely adopted a beta-sheet conformation on oxidized membranes, and the plane of the beta-sheet was oriented parallel to the plane of the membrane. These results indicate that oxidatively damaged phospholipid membranes promote beta-sheet formation by amyloid beta-proteins, and they suggest a possible role for lipid peroxidation in the pathogenesis of Alzheimer's Disease.     

Protease So from Escherichia coli preferentially degrades oxidatively damaged glutamine synthetase

After oxidative damage (e.g. induced with iron, ascorbate, and oxygen), the inactivated glutamine synthetase is selectively hydrolyzed in extracts of Escherichia coli. We therefore tested if glutamine synthetase treated with this system is hydrolyzed preferentially by any of the known E. coli proteases. Protease So, a cytoplasmic serine protease, was found to degrade the oxidized form of glutamine synthetase to acid-soluble peptides 5-10 times faster than the native glutamine synthetase. Degradation of the oxidized glutamine synthetase was inhibited by EDTA and stimulated 5-10-fold by Mg2+, Ca2+, or Mn2+, even though casein hydrolysis by protease So is not affected by divalent cations. Apparently, these cations affect the conformation of this substrate, making it more susceptible to proteolytic attack. Protease Re, another cytoplasmic protease, also degrades preferentially the oxidized form of glutamine synthetase and seems to correspond to the glutamine synthetase-degrading activity recently described by Roseman and Levine [1987) J. Biol. Chem. 262, 2101-2110). However, it is much less active in this reaction than protease So. No other soluble E. coli protease, including Do, Ci, Mi, Fa, Pi, or the ATP-dependent proteases Ti and La (the lon product), appears to degrade this oxidized protein. These results suggest that protease So participates in the hydrolysis of oxidatively damaged proteins and that E. coli has multiple systems for degrading different types of aberrant proteins.     

Characterization of two oxidatively modified phospholipids in mixed monolayers with DPPC

The properties of two oxidatively modified phospholipids viz. 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) and 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), were investigated using a Langmuir balance, recording force-area (pi-A) isotherms and surface potential psi. In mixed monolayers with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) a progressive disappearance of the liquid expanded-liquid condensed transition and film expansion was observed with increasing content of the oxidized phospholipids. The above is in agreement with fluorescence microscopy of the monolayers, which revealed an increase in the liquid expanded region of DPPC monolayers. At a critical pressure pi(s) approximately 42 mN/m both Poxo- and PazePC induced a deflection in the pi-A isotherms, which could be rationalized in terms of reorientation of the oxidatively modified acyl chains into aqueous phase (adaptation of the so-called extended conformation), followed upon further film compression by solubilization of the oxidized phospholipids into the aqueous phase. Surface potential displayed a discontinuity at the same value of area/molecule, corresponding to the loss of the oxidized phospholipids from the monolayers. Our data support the view that lipid oxidation modifies both the small-scale structural dynamics of biological membranes as well as their more macroscopic lateral organization. Accordingly, oxidatively modified lipids can be expected to influence the organization and functions of membrane associated proteins.     

Release of phospholipids from liposomal model membrane damaged by antibody and complement

When liposomal membranes were attacked by antibody and complement, enzymatic degradation of membrane phospholipids was not observed. However, intact membrane phospholipids were released into the surrounding medium in amounts beyond that expected from nonspecific protein-phospholipid interaction. On the other hand, when liposomal membranes were treated with phospholipase A purified from venom of "Habu" snake (Trimeresurus flavoviridis), trapped glucose marker was easily released, but phospholipids or their degradation products were not released into the medium and remained in the membrane structure.     

Augmentation of macrophage recognition of oxidatively damaged erythrocytes by substratum-bound fibronectin and macrophage surface fibronectin.

Thioglycollate-induced mouse peritoneal macrophages plated on a coverglass bind oxidized mouse erythrocytes in the absence of serum. Macrophages plated on a coverglass pre-coated with fibronectin (FN) were more active in binding of the oxidized erythrocytes. This effect of FN-coated coverglass was due to specific binding of an RGD-containing sequence of FN to FN-receptors on the macrophage, since GRGDSP hexapeptide in solution inhibited this effect, and GRGDSP-coated coverglass exhibited the same effect as FN-coated coverglass. Removal of FN originally present on the macrophage surface by trypsinization, prior to attachment to the coverglass, resulted in diminution of their ability of recognition of the oxidized erythrocytes, but the diminished ability was restored when the trypsinized macrophages were plated on a FN-coated coverglass, indicating that the cell surface FN is required for the macrophage recognition. Attachment to the coverglass was necessary for the cell surface FN to be effective. These results suggest that solid-phase FN, produced either by deposition of soluble FN to substratum or attachment of macrophage surface FN to substratum, activates the macrophages and augments their ability to recognize the oxidized erythrocytes.     

Involvement of calcium signaling in the fibronectin-stimulated macrophage recognition of oxidatively damaged erythrocytes

Macrophages recognize oxidatively damaged autologous erythrocytes, and cell surface fibronectin of macrophages enhances the recognition (Beppu et al., FEBS Lett. 295 (1991) 135-140). In the present study, mechanisms of enhanced macrophage recognition of oxidatively damaged erythrocytes by fibronectin were investigated. Monolayers of thioglycollate-induced mouse peritoneal macrophages with cell surface fibronectin recognized autologous erythrocytes oxidized with an iron catalyst ADP/Fe(3+). The macrophage recognition of the oxidized erythrocytes was inhibited partially by pretreatment of the macrophage monolayers with a Ca(2+) channel blocker (diltiazem), calmodulin inhibitors (W-7, trifluoperazine, chlorpromazine and dibucaine), an inhibitor of myosin light chain kinase (ML-9), a microfilament formation inhibitor (cytochalasin B), phospholipase A(2) inhibitors (4-bromophenacyl bromide, mepacrine) and cyclooxygenase inhibitors (indomethacin and aspirin). Monolayers of macrophages depleted of fibronectin by trypsinization lost the ability of recognizing oxidized erythrocytes, but acquired the ability when stimulated with a fibronectin-coated coverslip. The recognition of fibronectin-stimulated trypsinized macrophages was also inhibited by the above inhibitors. On treatment with Ca ionophore A23187, trypsinized macrophages acquired the ability to recognize oxidized erythrocytes. The recognition of Ca ionophore-stimulated trypsinized macrophages was inhibited by the above inhibitors except the Ca(2+) channel blocker. These results indicate that the Ca(2+) signaling including Ca(2+) influx, calmodulin activation and myosin light chain phosphorylation are involved in the fibronectin stimulation of the recognition of macrophages for oxidized erythrocytes. Involvement of microfilament formation and arachidonate cascade in the fibronectin stimulation was also suggested.