Ultrasensitive and specific detection methods for exocylic DNA adducts: Markers for lipid peroxidation and oxidative stress
Introduction
Environmental agents such as ultraviolet light, ionizing radiation, chemical carcinogens and cigarette smoke can induce oxidative stress in organisms. Most cells possess antioxidant defence systems composed of two components: the antioxidant enzymes consisting of superoxide dismutase, catalase and glutathione peroxidase, and the low molecular weight antioxidants such as vitamins A and E, ascorbate, glutathione and thioredoxin. These substances are the body's natural defence against endogenously generated reactive oxygen species (ROS) and other free radicals as well as ROS generated by exogenous environmental factors. Oxidative stress occurs when the production of ROS exceeds the body's natural antioxidant defence mechanisms, causing damage to macromolecules such as DNA, proteins and lipids.
Identification of endogenous sources for DNA damage and the resulting oxidative modification of cellular components will give new insights into the disease process as well as provide a better basis for chemoprevention of ROS-induced chronic degenerative diseases. Do we have sensitive and specific markers for oxidative DNA-base damage that we can apply in molecular epidemiological studies to humans? Many current detection methods are too insensitive for human biomonitoring and one of the most widely used marker 8-oxo-7,8-dihydro-2′-deoxy-guanosine (8-oxodG) produced results that varied over several orders of magnitude, likely due to its artefactual formation (Collins et al., 1996). Another potential problem exists for the use of 8-oxodG as a marker of oxidative stress in chronic inflammation/infection due to its degradation to ring opened products by peroxynitrate (generated from NO and O2−) formed concurrently as a result of induced iNOS (Uppu et al., 1996, Niles et al., 1999). Therefore, 8-oxo-dG may not always be a good cellular marker for the oxidative stress status alone, especially for evaluation of DNA-damage in chronic inflammation/infection.
Increased oxidative stress and lipid peroxidation (LPO) causing DNA damage and disturbance of cell signalling pathways, are being implicated in human cancers, neurodegenerative diseases and ageing processes. A role for LPO in tumor promotion and progression has been long postulated, but only recently it has been demonstrated that DNA adducts can be formed from reactive LPO products (reviewed in Bartsch, 1999). Persistent oxidative stress is also more and more recognized as a driving force to malignancy applicable to many epithelial tumors and it involves many steps of the multistage carcinogenesis process. As a cause cellular overproduction of ROS and reactive nitrogen species (RNS) are implicated. They are produced not only by normal physiological processes, but also in excess amounts during various pathophysiological conditions. ROS are generated as a result of energy production from mitochondria (from the electron transport chain), and of detoxification reactions carried out by the cytochrome P-450 system. Also as a part of the host antimicrobial and antiviral defence system a burst of ROS/RNS is produced during chronic infections and inflammatory processes, many of which are recognized risk factors for human cancers (Ohshima and Bartsch, 1994, Liu and Hotchkiss, 1995).
The oxidation of lipids by ROS and radicals, notably LPO of polyunsaturated fatty acids (PUFA), results in reactive products such as croton aldehyde, malondialdehyde and 4-hydroxyalkenals. These intermediates can react with DNA bases in vitro and in vivo to form exocyclic DNA adducts. Several have been characterized as propano and etheno DNA-base adducts. The evidence (summarized in Chung et al., 1996, Bartsch, 1999) indicates that etheno adducts are generated by reaction of DNA bases with lipid peroxidation products, as depicted in Fig. 1, involving the following steps: LPO produces fatty acid hydroperoxides and then the reactive trans-4-hydroxy-2-nonenal, a major product in vivo, which can be oxidized by hydrogen peroxide or fatty acid hydroperoxides to form its epoxide intermediate. This bifunctional agent attacks the nitrogen atom in DNA bases to form the etheno ring in cytidine, adenosine and guanosine. In support of this mechanism, etheno adducts were formed after peroxidation of arachidonic acid and microsomal membranes in the presence of iron(II) ions or cumene hydroperoxide (El Ghissassi et al., 1995). Three etheno adducts 1,N6-ethenodeoxyadenosine (εdA) 3, N4-ethenodeoxycytidine (εdC) (shown in Fig. 1) and N2,3-ethenodeoxyguanosine (εdG) have been detected in vivo. We have, therefore, concentrated on more stable secondary oxidation products as more useful markers of oxidative stress-derived DNA damage, in particular the promutagenic etheno (ε)-DNA adducts. Etheno adducts are also formed from the carcinogens vinyl chloride and urethane (Fernando et al., 1996) via their reactive oxirane intermediates. Thus various exogenous and endogenous sources can contribute to the mutagen burden imposed by these miscoding lesions.
Recently developed sensitive and specific methods allowed to detect these ε-adducts in vivo and to study their role in experimental and human carcinogenesis. The promutagenic ε-DNA modifications in tissues can be quantitated by an ultrasensitive immunoaffinity/32P-postlabelling procedure or visualized by immunohistochemistry. ε-Nucleosides (εdA) can be quantified in urine by an immunoaffinity-HPLC-fluorescence method. Highly variable background levels of ε-adducts were detected in tissues from unexposed humans and rodents, suggesting an endogenous pathway of ε-adduct formation by reaction of trans-4-hydroxy-2-nonenal (via its 2,3-epoxide) with DNA bases. Several known human cancer risk factors increased the level of ε-DNA lesions in human study subjects. Therefore, biomonitoring of exocyclic DNA adducts in man should offer new tools in cancer etiology research and in verifying the efficacy of chemopreventive agents in reducing endogenous DNA damage and cancer risk. This review will focus on newly developed detection methods and some applications of etheno adducts as lead markers for oxidative stress- and LPO-induced DNA damage in human and experimental systems. For a more comprehensive treatise the reader is referred to a recent compilation of this research area (Singer and Bartsch, 1999).
Section snippets
DNA-adduct analysis in tissues
εdA and εdC can be analyzed in DNA of various human and animal tissues by immunoaffinity/32P-postlabelling method (Nair et al., 1995). In brief, ∼25 μg of DNA is hydrolyzed to nucleotide-3′-monophosphate, using micrococcal nuclease and spleen phosphodiesterase. Normal nucleotides are quantitated by HPLC, and the adducts are enriched on immunoaffinity columns prepared from the monoclonal antibodies EM-A-1 for (εdA) and EM-C-1 for (εdC). The antibodies were obtained through a collaborative study
Existence of background levels of etheno-DNA adducts in tissues from unexposed rodents and human
We analyzed the etheno adducts εdC and εdA by a highly specific, sensitive method involving immunoaffinity chromatography coupled with 32P-postlabelling (Nair et al., 1995). Use of this method unambiguously and quantitatively revealed the existence of background levels of εdA and εdC in tissues from unexposed rodents and humans (Bartsch et al., 1994, Nair et al., 1995, Nair et al., 1999a).
Analysis of liver DNA samples from humans with unknown exposure showed the presence of εdA and εdC residues
Perspectives
With the advent of ultrasensitive, specific detection methods for etheno-DNA adducts in human tissues and cells, new insights can be gained into the mechanisms involved in human cancers with poorly defined aetiology and pathogenesis. Non-invasive methods for monitoring excreted etheno-deoxynucleosides in human urine as potential markers for LPO-related DNA damage occurring in the body, such as the HPLC-fluorescence method developed for εdA in our lab, will obviously be helpful. Most
Acknowledgements
The authors' research in this area was in part supported by EU contract ENV4-CT97-0505. The authors wish to acknowledge the work contributed by A. Barbin and K. Schmid and the collaboration with H. G. Beger, M. Mutanen, M. Nagao, D.H. Phillips, S. Tannenbaum, C. Vaca, G. Winde and G. Wogan. Dr P. Lorenz, University of Essen, Essen Germany is thanked for providing the Mab used in this study. C. Ditrich and I. Hofmann is thanked for skilled technical assistance and S. Fuladdjusch for excellent
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