2. Sensitivity and specificity
of techniques for
the identification of biomarkers
2.1. DNA adducts
2.1.1. Introduction
Peter B. Farmer
University of Leicester, Leicester, UK
The measurement of DNA adducts is one of the most important biomarkers for exposure to genotoxic carcinogens, as it gives an indication of the biologically effective dose of the carcinogen that has reached the tissue DNA under study. Additionally the extent of DNA adduct formation may be indicative of the risk associated with the exposure. For example, the pioneering study by Ross et al. (1992) [1] showed that adduct formation from dietary intake of aflatoxin B1 was statistically related to the subsequent incidence of hepato-cellular carcinoma. A recent meta-analysis by Peluso et al. (2005) [2] showed that leuko-cyte DNA adducts were associated with the subsequent risk of lung cancer, the associa-tion being stronger in never-smokers.
The assay methods available for DNA adducts vary greatly in their sensitivity and selectivity, the amount of DNA that is required, and the window of exposure for which the methods can be applied (see Section 1; Farmer, 2004 [3]). Sensitivities achieved are normally in the range 1 adduct/109nucleotides to 1 adduct/108nucleotides, sufficient
to give useful information on human environmental or dietary exposures. Although many of these methods have been validated in animal models where single compound exposures have been used, human exposure is to complex mixtures of genotoxic compounds from a variety of sources, and the challenge for the successful use of these biomarkers is therefore much greater. Some of the most commonly used assays [e.g. 32P-postlabelling (Section 2.1.2.), and some immunoassays (Section 2.1.4.)] are
capable of measuring mixtures of DNA adducts, and others [e.g. mass spectrometry (Section 2.1.3.)] hold particular advantage for individual adduct determination. Comparison of biomarker methods for a particular adduct is only possible if one knows the efficiency of recovery of the adduct from DNA and the accuracy of each quantitative procedure, which is not always the case. Endeavours to undertake extensive procedures for prepurification of DNA to isolate a specific adduct normally require large amounts of DNA and are therefore not amenable to the screening of large numbers of biological
David Phillips 18
samples. There is no perfect approach for the measurement of DNA adducts in humans, and the procedures should be selected on a ‘case-by-case’ basis. An understanding of the nature of the exposure and the chemistry of the adduct in question is essential before choosing the experimental approach for adduct quantification.
References
1. Ross RK, Yuan J-M, Yu MC, Wogan GN, Qian G-S, Tu J-T, et al. Urinary aflatoxin biomarkers and risk of hepatocellular carcinoma. Lancet 1992;339:943–6.
2. Peluso M, Munnia A, Hoek G, Krzyzanowski M, Veglia F, Airoldi L, et al. DNA adducts and lung cancer risk: A prospective study. Cancer Res 2005;65:8042–8.
3. Farmer PB. Exposure biomarkers for the study of toxicological impact on carcinogenic processes. In: Buffler P, Rice J, Bird M, Boffetta P, editors. Mechanisms of carcinogenesis. Contributions of molecular epidemiology. IARC Scientific Publication No. 157. Lyon: IARC Press; 2004. p. 71–90.
2.1.2. 32P-Postlabelling
David Phillips
Institute of Cancer Research, London, UK
The 32P-postlabelling assay has been in use for over 20 years [1]. It is a highly sensitive
technique whereby modified bases in DNA are tagged with a radioactive group and detected by means of their radioactive decay. The use of a high specific activity radioactive isotope, namely phosphorus-32, permits the assay to detect DNA adducts formed in human tissues as a result of either occupational or environmental exposure. The method has been widely used and reviewed [2–5].
For most practical purposes the assay consists of four main steps. (i) DNA digestion; (ii) a procedure that isolates or selects the adducts for preferential labelling; (iii) polynucleotide kinase (PNK)-mediated phosphorylation of the adducts using [γ-32P]ATP; and (iv) chromatographic resolution of the labelled adducts and their detection
and quantitation by means of their 32P-decay.
Polynucleotide kinase will mediate the phosphorylation of the 5’-position on deoxyribose provided the 3’-position is phosphorylated, i.e. if the substrate is a nucleotide or polynucleotide. Thus stage 1, DNA digestion, usually involves enzymatic digestion with micrococcal nuclease and spleen phosphodiesterase. This generates nucleoside 3’-phosphates. After postlabelling, 3’,5’-bisphosphates, containing the radiolabel, are generated. These can be resolved and detected in this form; alternatively further digestion with nuclease P1 converts them to labelled nucleoside 5’-monophosphates [6]. A different digestion method involves using nuclease P1 and prostatic acid phosphatase, which
19
results in normal nucleosides and adducted dinucleotides due to the resistance of modified nucleotides to complete digestion [7]. After postlabelling, further digestion by venom phosphodiesterase cleaves the phosphodiester bond to generate the labelled adduct as a modified nucleoside 5’-monophosphate.
Strategies that avoid the labelling of non-adducted nucleotides can greatly enhance the sensitivity of the method. The two most commonly used are butanol extraction [8], whereby adducted nucleotides are selectively isolated by solvent extraction and then subjected to 32P-labelling, and nuclease P1 digestion [9], whereby normal nucleoside
3’-monophosphates are converted to nucleosides (which are not substrates for PNK) under conditions in which some, but not all, classes of adducts are resistant to digestion (and thus remain substrates for the labelling reaction).
Other procedures for sensitivity enhancement include extraction of adducts prior to labelling using solid-phase adsorption including high performance liquid chromato-graphy (HPLC) [10], small cartridge columns [11,12] or immunoaffinity columns containing antibodies to the adducts of interest [13,14].
Resolution and detection of adducts is commonly carried out by thin-layer chromatography (TLC) because: (i) this is sensitive, allowing visualisation of adducts as radioactive spots or ‘zones’ using autoradiography or an equivalent electronic radioactivity imaging system; (ii) many samples can be analysed in parallel; and (iii) cross-contamination of samples can be avoided. Nevertheless, the procedure is time consuming, labour intensive and has low resolution. Alternatively, HPLC separation can be used [15], although this requires samples to be analysed in series, it is less sensitive and great care is needed to avoid cross-contamination. There is, at present, considerable scope for improving the assay through the coordinated development of higher throughput, semi-automated procedures. Such developments would enhance considerably the application of the technique to large-scale human biomonitoring studies (see below).
The sensitivity with which different DNA adducts can be detected by 32P-postlabelling is variable, but optimally limits of detection as low as 1 adduct
in 1010nucleotides can be achieved in a sample of around 10 µg. More routinely, limits
of sensitivity of at least 1 in 109nucleotides are achieved for bulky adducts.
Biomonitoring studies using 32P-postlabelling are now very numerous. Many of them
reveal complex patterns of DNA adducts that are broadly characteristic of the patterns formed in experimental systems by complex mixtures of bulky carcinogens, such as polycyclic aromatic hydrocarbons (PAHs), although the exact nature of much of the DNA damage that is being detected by 32P-postlabelling in these circumstances is still
relatively uncharacterised. The definition of what constitutes a ‘bulky adduct’ is also open to variable interpretation, but in general it is taken to include aromatic moieties with two or more aromatic rings, and some large extended non-aromatic or aliphatic structures. Quantitative differences between environmentally exposed and unexposed groups have been noted in many studies [16], as have differences between the adduct levels detected in many tissues of smokers relative to non-smokers [17]. In many cases, Biomarker identification techniques: 32P-Postlabelling
David Phillips 20
the potential of the technique is restricted by its being so labour intensive, inhibiting its application to truly large-scale human biomonitoring studies.
There have been a few small-scale interlaboratory trials to compare 32P-postlabelling
results between laboratories [18–20] and one larger-scale trial [21]. Although some external adduct standards have been prepared [21,22], these are not used routinely to normalise results obtained with human samples. There is undoubtedly a need for more standardised protocols to allow comparisons between results obtained in different laboratories, a process that was started in the large interlaboratory trial, but by no means completed [21]. Nevertheless, 32P-postlabelling analysis is the most versatile
of the methods available for monitoring DNA adduct formation in humans and it has the potential to provide significant insight into the etiology of human cancer, as well as in monitoring human exposure to known environmental sources of carcinogens.
References
1. Randerath K, Randerath E. Postlabelling methods — an historical review. In: Phillips DH, Castegnaro M, Bartsch, H, editors. Postlabelling methods for detection of DNA adducts. Lyon: International Agency for Research on Cancer; 1993. p. 3–9.
2. Beach AC, Gupta RC. Human biomonitoring and the 32P-postlabelling assay. Carcinogenesis 1992;13:1053–74.
3. Strickland PT, Routledge MN, Dipple A. Methodologies for measuring carcinogen adducts in humans. Cancer Epidemiol Biom Prev 1993,2:607–19.
4. Randerath K, Randerath E. 32P-Postlabelling methods for DNA adduct detection: overview and critical evaluation. Drug Metab Rev 1994;26:67–85.
5. Phillips DH. Detection of DNA modifications by the 32P-postlabelling assay. Mutat Res 1997;378:1–12.
6. Pfau W, Brockstedt U, Söhren K, Marquardt H. 32P-Post-labelling analysis of DNA adducts formed by food-derived heterocyclic amines: evidence for incomplete hydrolysis and a procedure for adduct pattern simplification. Carcinogenesis 1994;15:877–82.
7. Randerath K, Randerath E, Danna TF, van Golen KL, Putman KL. A new sensitive 32P-postlabeling assay based on the specific enzymatic conversion of bulky DNA lesions to radio-labeled dinucleotides and nucleoside 5’-monophosphates. Carcinogenesis 1998;10:1231–9. 8. Gupta RC. Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen: DNA
adducts. Cancer Res 1985;45:5656–62.
9. Reddy MV, Randerath K. Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis 1986;7:1543–51.
10. Dunn BP, San RHC. HPLC enrichment of hydrophobic DNA adducts for enhanced sensitivity of 32P-postlabeling analysis. Carcinogenesis 1988;9:1055–60.
11. Turesky RJ, Markovic J. DNA adduct formation of the food carcinogen 2-amino-3-methylimi-dazo[4,5-f]quinoline at the C-8 and N2atoms of guanine. Chem Res Toxicol 1994;7:752–61. 12. Phillips DH, Carmichael PL, Hewer A, Cole KJ, Hardcastle IR, Poon GK, et al. Activation
of tamoxifen and its metabolite α-hydroxytamoxifen to DNA-binding products: comparisons between human, rat and mouse hepatocytes. Carcinogenesis 1996;17:88–94.
21 Biomarker identification techniques: 32P-HPLC
13. Cooper DP, Griffin KA, Povey AC. Immunoaffinity purification combined with 32P-postlabelling for the detection of O6-methylguanine in DNA from human tissues. Carcinogenesis 1992;13:469–75. 14. Nair J, Barbin A, Guichard Y, Bartsch H. 1,N6-Ethenodeoxyadenosine and 3,N4
-ethenodeoxy-cytidine in liver DNA from humans and untreated rodents detected by immuno-affinity/32P-postlabelling. Carcinogenesis 1995;16:613–7.
15. Möller L, Zeisig M, Vodicka P. Optimization of an HPLC method for analyses of 32P-postlabeled DNA adducts. Carcinogenesis 1993;14:1343–8.
16. Phillips DH. Macromolecular adducts as biomarkers of human exposure to polycyclic aromatic hydrocarbons. In: Luch A, editor. The carcinogenic effects of polycyclic aromatic hydrocarbons. London: Imperial College Press; 2005; p. 137–69.
17. Phillips DH. Smoking-related DNA and protein adducts in human tissues. Carcino-genesis 2002;23:1979–2004.
18. Hemminki K, Grzybowska E, Chorazy M, Twardowska-Saucha K, Sroczynski JW, Putman KL, et al. DNA adducts in humans environmentally exposed to aromatic compounds in an industrial area of Poland. Carcinogenesis 1990;11:1229–31.
19. Savela K, Hemminki K, Hewer A, Phillips DH, Putman KL, Randerath K. Interlaboratory comparison of the 32P-postlabelling assay for aromatic DNA adducts in white blood cells of iron foundry workers. Mutat Res 1989;224:485–92.
20. Phillips DH, Castegnaro M. Results of an interlaboratory trial of 32P-postlabelling. In: Phillips DH, Castegnaro M, Bartsch H, editors. Postlabelling methods for detection of DNA damage. Lyon: IARC; 1993, p. 35–49.
21. Phillips DH, Castegnaro M, on behalf of trial participants. Standardization and validation of DNA adduct postlabelling methods: report of interlaboratory trials and production of recommended protocols. Mutagenesis 1999;14:301–15.
22. Baan RA, Steenwinkel M-JST, van Asten S, Roggeband R, van Delft JHM. The use of benzo[a]pyrene diolepoxide-modified DNA standards for adduct quantification in 32 P-postla-belling to assess exposure to polycyclic aromatic hydrocarbons: application in a biomonitoring study. Mutat Res 1997;378:41–50.
Addendum
32P-HPLC
Lennart Möller
Leocordia AB, Selagarden, Stallarholmen, Sweden
32P-HPLC was developed in the early 1990s and proved to be somewhat less sensitive
than 32P-TLC with autoradiography. However, 32P-HPLC is usually faster, with better
separation, versatility and reproducibility compared with 32P-TLC [1]. 32P-HPLC, using
only HPLC for separation and online detection, in contrast to variants using TLC for pre-separation and radioactivity measurement on collected eluent fractions, has since been used to analyse many kinds of DNA-adduct samples, from in vitro experiments [2-4], in vivo experiments [5,6] and from humans, e.g. after exposure to
Lennart Möller 22
endogenous factors [7] and alkylation [8,9], specific environmental factors like ultra-violet radiation [10] and food-derived heterocyclic amines [11,12], more general en-vironmental exposures [13–15], specific occupational factors like 1,3-butadiene [16,17] and propylene oxide [18], more general occupational factors [19–22], and medical exposure such as from tamoxifen [23].
In addition there are studies in press that show DNA-adduct fingerprint changes in normal colonic epithelium during the disease process from controls to polyp patients to colon cancer cases [Möller et al. unpublished]. Further, application of 32P-HPLC
to human tissues showed different levels and patterns in different tissues of indivi-duals. The pancreas showed very high levels, suggesting a link to diabetes [Möller et al. unpublished]. Provocation of human lymphocytes with mutagens revealed a strong seasonal variation within the same individuals which was linked to a seasonal variation in CYP450 [Möller et al. unpublished]. Human lung tissue in different countries showed higher DNA-adduct levels than liver tissue, suggesting a connection with air pollutants [Möller et al. unpublished].
Reproducibility of retention times with the 32P-HPLC method is very good, enabling
comparison between chromatograms run many years apart by using endogenous peaks visible in all chromatograms for calibration. Quantitative reproducibility is somewhat less efficient owing to variations in enzyme batches and the [32P]ATP used for postlabelling.
References
1. Eriksson HL, Zeisig M, Ekstrom LG, Moller L. 32P-postlabeling of DNA adducts arising from complex mixtures: HPLC versus TLC separation applied to adducts from petroleum products. Arch Toxicol 2004;78:174–81.
2. Möller L, Zeisig M, Vodicka P. Optimization of an HPLC method for analyses of 32P-postlabeled DNA adducts. Carcinogenesis 1993;14:1343–8.
3. Zeisig M, Moller L. 32P-Postlabeling high-performance liquid chromatographic improvements to characterize DNA adduct stereoisomers from benzo[a]pyrene and benzo[c]phenanthrene, and to separate DNA adducts from 7,12-dimethylbenz[a]anthracene. J Chromatogr B Biomed Sci Appl 1997;691:341–50.
4. Zeisig M, Hofer T, Cadet J, Moller L. 32P-postlabeling high-performance liquid chromatography (32P-HPLC) adapted for analysis of 8-hydroxy-2’-deoxyguanosine. Carcinogenesis 1999;20:1241–5. 5. Möller L, Zeisig M. DNA adduct formation after oral administration of 2-nitrofluorene
and N-acetyl-2-aminofluorene, analyzed by 32P-TLC and 32P-HPLC. Carcinogenesis 1993;14:53–9. 6. Möller L., Zeisig M, Midtvedt T, Gustafsson JÅ. Intestinal microflora enhances formation
of DNA adducts following administration of 2-NF and 2-AAF. Carcinogenesis 1994;15:857–61. 7. Yang K, Fang JL, Hemminki K. Abundant lipophilic DNA adducts in human tissues. Mutat
Res 1998;422:285–95.
8. Kim DY, Cho MH, Yang HK, Hemminki K, Kim JP, Jang JJ, et al. Detection of methylation damage in DNA of gastric cancer tissues using 32
P-postlabelling assay. Jpn J Cancer Res 1999;90:1104–8.
23 Biomarker identification techniques: 32P-HPLC
9. Zhao C, Hemminki K. The in vivo levels of DNA alkylation products in human lymphocytes are not age dependent: an assay of 7-methyl- and 7-(2-hydroxyethyl)-guanine DNA adducts. Carcinogenesis 2002;23:307–10.
10. Bykov VJ, Hemminki K. UV-induced photoproducts in human skin explants analysed by TLC and HPLC-radioactivity detection. Carcinogenesis 1995;16:3015–9.
11. Banaszewski J, Szmeja Z, Szyfter W, Szyfter K, Baranczewski P, et al. Analysis of aromatic DNA adducts in laryngeal biopsies. Eur Arch Otorhinolaryngol 2000;257:149–53.
12. Baranczewski P, Möller L. Relationship between content and activity of cytochrome P450 and induction of heterocyclic amine DNA adducts in human liver samples in vivo and in vitro. Cancer Epidemiol Biomarkers Prev 2004;13:1071–8.
13. Möller L, Grzybowska E, Zeisig M, Cimander B, Hemminki K, Chorazy M. Seasonal variation of DNA adduct pattern in human lymphocytes analyzed by 32P-HPLC. Carcino-genesis 1996;17:61–6.
14. Carlberg CE, Möller L, Paakki P, Kantola M, Stockmann H, Purkunen R, et al. DNA adducts in human placenta as biomarkers for environmental pollution, analysed by the 32P-HPLC method. Biomarkers 2000;5:182–92.
15. Jaloszynski P, Jaruga P, Olinski R, Biczysko W, Szyfter W, Nagy E, et al. Oxidative DNA base modifications and polycyclic aromatic hydrocarbon DNA adducts in squamous cell carcinoma of larynx. Free Radic Res 2003;37:231–40.
16. Zhao C, Koskinen M, Hemminki K. 32P-postlabelling of N6-adenine adducts of epoxybutanediol in vivo after 1,3-butadiene exposure. Toxicol Lett 1998;102–103:591–4.
17. Zhao C, Vodicka P, Sram RJ, Hemminki K. Human DNA adducts of 1,3-butadiene, an important environmental carcinogen. Carcinogenesis 2000;21:107–11.
18. Czene K, Osterman-Golkar S, Yun X, Li G, Zhao F, Perez HL, et al. Analysis of DNA and hemo-globin adducts and sister chromatid exchanges in a human population occupationally exposed to propylene oxide: a pilot study. Cancer Epidemiol Biomarkers Prev 2002;11:315–8.
19. Carstensen U, Yang K, Levin JO, Ostman C, Nilsson T, Hemminki K, et al. Genotoxic exposures of potroom workers. Scand J Work Environ Health 1999;25:24–32.
20. Plna K, Osterman-Golkar S, Nogradi E, Segerback D. 32P-post-labelling of 7-(3-chloro-2-hydroxy-propyl)guanine in white blood cells of workers occupationally exposed to epichlorohydrin. Carcinogenesis 2000;21:275–80.
21. Sram RJ, Binkova B. Molecular epidemiology studies on occupational and environmental exposure to mutagens and carcinogens, 1997–1999. Environ Health Perspect 2000;108 (Suppl 1):57–70.
22. Tuominen R, Baranczewski P, Warholm M, Hagmar L, Möller L, Rannug A. Susceptibility factors and DNA adducts in peripheral blood mononuclear cells of aluminium smelter workers exposed to polycyclic aromatic hydrocarbons. Arch Toxicol 2002;76:178–86.
23. Hemminki K, Rajaniemi H, Lindahl B, Moberger B. Tamoxifen-induced DNA adducts in endometrial samples from breast cancer patients. Cancer Res 1996;56:4374–7.
Raj Singh 24
2.1.3. Mass spectrometric detection of DNA adducts
Raj Singh
University of Leicester, Leicester, UK
The use of mass spectrometry for the detection of DNA adducts has been recently reviewed by Singh and Farmer, with particular reference to liquid chromato-graphy-electrospray ionization-mass spectrometry (ESI-LC-MS) [1]. Further compre-hensive reviews describing the application of liquid chromatography coupled to mass spectrometry for the study of DNA adducts have been published by Esmans et al. (1998) Andrews et al. (1999), Doerge et al. (2002), Koc and Swenberg (2002) and Watson et al. (2003) [2–6]. Traditionally the role of mass spectrometry in the determination of DNA adducts has been limited to providing information for the identification of new DNA adducts, or for the structural characterisation of DNA adduct standards that have been utilised to determine adduct levels by other detection methods, such as 32P-postlabelling, described in Section 2.1.2. [7]. However, in recent times, owing
to technological advances in instrumentation, mass spectrometry has become a viable method for the quantitation of DNA adduct levels in animal and human samples obtained after exposure to exogenous and endogenous genotoxic compounds.
Accelerator mass spectrometry, which measures isotope ratios, represents the most sensitive analytical method so far available for detecting DNA adducts, with a limit of de-tection that may be as low as 1 adduct per 1012unmodified DNA bases [8]. For example,
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) DNA adducts at a level of 30–130 adducts per 1012unmodified DNA bases have been detected in normal human
colon tissue after exposure to dietary-relevant doses of the carcinogen [9]. The main limitation of the technique is that it depends on the presence of an isotope such as 14C
or 3H in the molecule of interest, which means that at present its application is restricted
to experimental systems where labelled compounds may be used. Numerous DNA adducts have been studied using gas chromatography-electron capture-negative chemical ionization-mass spectrometry (GC-EC-NCI-MS), examples of which include malondi-aldehyde-guanine, N-7-(2-hydroxyethyl)guanine, N2,3-ethenoguanine, 1,N2
-etheno-guanine and 1,N6-ethenoadenine [10–13]. Gas chromatography-mass spectrometry has
also been used to study DNA adducts that are derived from carcinogens normally present in tobacco smoke, such as 4-aminobiphenyl (4-ABP) and 4-(methylnitrosoamine)-1-(3--pyridyl)-1-butanone (NNK), as well as in the diet, such as PhIP [14–16]. Furthermore GC-MS has been used to demonstrate the presence of benzo[a]pyrene DNA adducts in human placental tissue [17]. The main advantage of gas chromatography is greater peak resolution compared with conventional liquid chromatography. The disadvantage is that only non-polar/volatile compounds can be analysed, and in the case of the majority of DNA adducts, which are non-volatile and/or polar, there is a requirement for derivatization at high temperature prior to analysis. The development of the electro-spray ionization source has meant that polar compounds can be directly analysed
25
without the need for derivatization, allowing the coupling of liquid chromatography to the mass spectrometer.
The process of electrospray ionization occurs at atmospheric pressure, resulting in negligible dissociation of the molecule ion into fragment ions, and is termed a soft ioniza-tion technique. Comprehensive reviews explaining the electrospray ionizaioniza-tion process in detail have been published by Gaskell (1997) and by Bruins (1998) [18,19]. Electrospray ionization is the main choice of ionization technique for studying DNA adducts, although atmospheric pressure chemical ionization has also been employed for their detection, albeit to a much lesser extent [20,21]. The most common design of instrument used for DNA adduct analysis is a triple quadrupole which allows for tandem mass spectrometry (MS/MS) to be performed. The mass spectrometer consists of the first (Q1) and third (Q3) quadrupoles which are the mass analysers (filters) separated by the second (q2) quadrupole called the collision cell. Analyte ions undergo collision induced dissociation (CID) in the collision cell, which is filled with an inert gas (normally argon) [1]. Time of flight instruments coupled with a quadrupole analyser (Q-TOF) have also been used to detect DNA adducts. The use of TOF instruments has been limited to providing high resolution data (accurate mass data) for the characterisation of DNA adducts, for example the adducts formed in the lung tissue of mice following exposure to asphalt fumes [22].
The two main detection methods used for determination of DNA adducts are single ion monitoring (SIM) and selected reaction monitoring (SRM). For SIM the first quadrupole (Q1) is set to pass only the ions of interest to the detector. In contrast SRM involves the selection of a precursor ion of the compound of interest in the first quadrupole (Q1), which undergoes transition in the collision cell (q2) by CID to a specific product ion, which is then determined by the third quadrupole (Q3). Opti-mum sensitivity and specificity for the quantitative analysis of DNA adducts is obtained by using SRM where a unique and abundant product ion is detected following CID [1]. Another tandem mass spectrometry detection mode is constant neutral loss (CNL), which is used primarily to screen for specific compounds that share a common CID pathway with the loss of a specific neutral moiety or functional group. CNL is applicable to the study of 2’-deoxynucleoside DNA adducts since they share a common CID pathway resulting in the loss of 2’-deoxyribose as a neutral moiety [23,24]. In terms of the analysis of DNA adducts by LC-MS the method of analysis can be applied to adducted purine or pyrimidine bases, 2’-deoxynucleosides as well as 2’-deoxy-nucleotides. Numerous studies have been published of the detection and quantification of DNA adducts ranging from small or polar adducts to bulky or non-polar adducts using ESI-LC-MS, further details of which can be found in the review by Singh and Farmer [1]. The main advantage mass spectrometry has to offer compared with other analytical methods is that it provides information about the structural identity of the compound under investigation, thus confirming that the correct compound is being studied. Accurate quantitation of DNA adduct levels is achieved by the use of stable isotope internal standards. These standards are identical in every respect chromatographically to the DNA adduct of interest but differ in mass and hence can only be differentiated Biomarker identification techniques: Mass spectrometric detection of DNA adducts
Raj Singh 26
by mass spectrometry. The stable isotope internal standards are normally added at the start of the procedure, thus accounting for any losses during sample manipulation and preparation. The stable isotope internal standards also account for any variations in the response of the mass spectrometer such as suppression of ionization due to matrix effects [1,25]. The typical detection limits of ESI-LC-MS that have been quoted for various DNA adducts range from 0.5–5 adducts per 108unmodified DNA bases. Analyses are typically
performed using 50–100 µg of DNA [26–28]. The results obtained using ESI-LC-MS have been compared with results obtained using other detection techniques such as 32
P-post-labelling and immunoassays. For example, Beland et al. [29] compared ESI-LC-MS,
32P-postlabelling and dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA)
for the determination of 4-ABP adduct levels in calf thymus DNA that was reacted in vitro, and in the liver DNA of mice treated with radiolabelled [3
H]N-hydroxy-4--aminobiphenyl. The results showed that 32P-postlabelling underestimated adduct levels
(3–5% relative level), DELFIA overestimated adduct levels (260–550% relative level) and that ESI-LC-MS gave levels of adducts that agreed most closely with those obtained for the specific binding of the 3H-radiolabel to the mouse liver DNA. Furthermore the authors
found that the LC-MS method provided the most accurate and precise results following statistical evaluation of the data when compared with the other two methods [29].
Further gains in the sensitivity of LC-MS for the detection of DNA adducts, particularly for the levels of adducts found in human samples, can be achieved by using capillary and nanocapillary LC (to replace the currently employed narrow bore and micro-bore LC) coupled to micro- or nanoelectrospray ionization mass spectrometry [30,31]. For example Ricicki et al. used capillary LC coupled to microelectrospray ionization-mass spectrometry for the quantitation of 4-ABP DNA adducts in human pancreas tissue using only 13.3 µg of the processed DNA sample for the analysis [32]. The coupling of capillary electrophoresis to mass spectrometry to study DNA adducts is reviewed in [33].
Mass spectrometry can also provide information regarding the site of the adducts within the DNA sequence. For example LC-ESI-MS/MS has been used to provide sequence information for numerous model studies where oligonucleotides have been adducted with genotoxic carcinogens in vitro [34,35]. The technique of matrix assisted laser desorption ionization (MALDI) has been used for DNA sequencing for the detection of mutations. Due to its wide mass range and ease of automation for high speed sample throughput a wide variety of approaches have been developed for genotyping [single nucleotide polymorphism (SNP) detection] and mutation detection using MALDI-MS. The use of mass spectrometry for genotyping has been reviewed by Jackson et al. (2000) and Edwards et al. (2005) [36,37].
Recently a LC-MS approach has been developed for the detection of mutations in short DNA fragments. Referred to as short oligonucleotide mass analysis (SOMA), this procedure involves polymerase chain reaction (PCR) amplification of the area of interest of the gene with primers containing a sequence for the restriction endonuclease BpmI, and digestion of the PCR product with BpmI, liberating short (< 20-mer) oligonucleotides
27 Biomarker identification techniques: Mass spectrometric detection of DNA adducts
that can be analysed by LC-ESI-MS/MS (or MALDI-TOF) [38]. This approach has been used for the detection of base changes in codon 249 of the p53 gene, the hepatitis B virus and codon 12 of the K-ras gene [39–41].
The technological advances in mass spectrometry are still on-going, for example improvements in instrumentation have resulted in increased efficiency of ionization, transmission and detection of ions. There seems to be no doubt that in future mass spectrometry will take on a larger role in DNA adduct determinations in human studies, which is highlighted by the increasing number of publications employing LC-MS for the detection of DNA adducts in human populations exposed to genotoxic carcinogens.
References
1. Singh R, Farmer PB. Liquid chromatography-electrospray ionization-mass spectrometry: the future of DNA adduct detection. Carcinogenesis 2006;27:178–96.
2. Esmans EL, Broes D, Hoes I, Lemiere F, Vanhoutte K. Liquid chromatography-mass spectro-metry in nucleoside, nucleotide and modified nucleotide characterization. J Chroma-togr A 1998;794:109–27.
3. Andrews CL, Vouros P, Harsch A. Analysis of DNA adducts using high-performance sepa-ration techniques coupled to electrospray ionization mass spectrometry. J Chroma-togr A 1999;856:515–26.
4. Doerge DR, Churchwell MI, Beland FA Analysis of DNA adducts from chemical carcinogens and lipid peroxidation using liquid chromatography and electrospray mass spectrometry. Environ Carcino Ecotox Revs 2002;C20:1–20.
5. Koc H, Swenberg JA. Applications of mass spectrometry for quantitation of DNA adducts. J Chromatogr B 2002;778:323–43.
6. Watson DG, Atsriku C, Oliveira EJ. Review role of liquid chromatography-mass spectrometry in the analysis of oxidation products and antioxidants in biological systems. Anal Chim Acta 2003;492:17–47.
7. Talaska G, Roh JH, Getek T. 32P-Postlabelling and mass spectrometric methods for analysis of bulky, polyaromatic carcinogen-DNA adducts in humans. J Chromatogr 1992;580:293–323. 8. Turteltaub KW, Dingley KH. Application of accelerated mass spectrometry (AMS) in DNA
adduct quantification and identification. Toxicol Lett 1998;102–103:435–9.
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26. Singh R, Kaur B, Farmer PB. Detection of DNA damage derived from a direct acting ethylating agent present in cigarette smoke by use of liquid chromatography-tandem mass spectrometry. Chem Res Toxicol 2005;18:249–56.
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32. Ricicki EM, Soglia JR, Teitel C, Kane R, Kadlubar F, Vouros P. Detection and quantifi-cation of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human pancreas tissue using capillary liquid chromatography-microelectrospray mass spectrometry. Chem Res Toxicol 2005;18:692–99.
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2.1.4. Other techniques to detect DNA adducts
Frederik-Jan van Schooten and Roger Godschalk Maastricht University, Maastricht, The Netherlands
The 32P-postlabelling assay is currently the most sensitive assay to detect DNA
modifi-cations and is therefore frequently applied to assess DNA adduct levels in both in vivo and in vitro experiments as well as in carcinogen-exposed humans. However, despite its high sensitivity and the requirement of small amounts of DNA, this assay also has its disad-vantages, including:
• the inability to identify DNA adduct types,
• the use of relatively high levels of radioactivity and thus need for specialised laboratory equipment and protection, and
• its time-consuming nature with limited possibilities to optimise the procedure further for high-throughput analysis of DNA samples.
Therefore, other techniques are in use to counteract the shortcomings of the 32
P-post-labelling assay. In this short overview, the following techniques will be discussed (classi-fied according to the principle of DNA-adduct detection):
• immunoassays,
• assays based on the fluorescent properties of the DNA adducts,
• assays based on fluorescence detection after fluorescent labelling of DNA adducts, • mass-spectrometric detection of DNA adducts, and
• other techniques. Immunoassays
The use of immunoassays in the detection and quantitation of DNA modifications is well reviewed [1–3]. The development of sensitive and simple immunoassays was made possible by the production of poly- and monoclonal antibodies against structural modifi-cations of DNA. The first antisera against modified DNA were raised against the alky-lation adducts O6-methyl- and O6-ethylguanine, 7-methylguanine, and N6-methyladenine
([4] and reviewed in [5]). For bulky chemical carcinogens, the first antibodies recognised DNA adducts formed by N-2-acetylaminofluorene [6]. Thereafter, numerous other antibodies were raised to various types of DNA modifications, including DNA adducts formed by benzo[a]pyrene-diol epoxide [7,8], 4-aminobiphenyl [9], aflatoxin B1 [5], alkylating compounds (methylating and ethylating agents) [5] and adducts formed by oxidative stress [such as malondialdehyde [10], etheno adducts [11] and 8-oxo-7,8--dihydro-2-deoxyguanosine (8-oxo-dG) [12]]. Although antibodies are thought to react specifically with certain DNA adducts, it appears that they may cross-react with different structurally similar DNA adducts. Therefore, they are considered to be class specific rather than adduct specific. Moreover, antibodies raised against highly modified DNA may have difficulty in recognising little-modified DNA, resulting in an underestimation of the actual adduct level in the unknown sample [8]. Therefore, it is advisable to prepare
31
calibration curves with modification levels that are similar to those expected in the samples. Antibodies were also successfully applied in purification steps with subsequent detection of DNA adducts by the ultrasensitive 32P-postlabelling assay (see Section 2.1.2.
for information on 32P-postlabelling). As a result, the specificity of antibodies can be
com-bined with the sensitivity of the 32P-postlabelling assay [13,14]. However, this application
of antibodies will not be discussed here.
Enzyme-linked immunosorbent assay
Although radioimmunoassays (RIAs) were initially applied to assess the relatively low DNA adduct levels [7] because these assays are highly sensitive and reproducible, they have generally been replaced by enzyme-linked immunosorbent assays (ELISAs), which do not require the use and handling of radioactive material, but are still sufficiently sensitive to detect DNA adducts in exposed humans. Different types of ELISA can be used for DNA-adduct detection. However, the most frequently applied version is the com-petitive ELISA (cELISA, e.g. for the detection of BPDE-dG) [1,8]. There are numerous variations of the ELISA using alkaline phosphatase or peroxidase-conjugated secondary antibodies for the detection of adduct-antibody interactions [1]. In this assay, carcinogen-modified DNA or monoadduct coupled to carrier protein is coated onto 96-microwell plates. Nonspecific binding of antibodies to the plate is subsequently blocked by incu-bation with a protein solution. A standard curve is generated by serial dilution of either the modified denatured DNA or monoadduct and mixing with diluted antibody. Samples with unknown levels of DNA adducts are tested in a similar way. Antibody binding to the DNA adduct coated on the plate is competed by DNA adducts in solution. After incubation and washing off of unbound material, bound primary antibody is quantitated with enzyme-conjugated secondary antisera, followed by the appropriate substrate [1]. With the highest affinity antibodies, 50% inhibition can be obtained with adduct levels in the femtomole range. With this level of sensitivity and the ability to assay 25 to 35 µg of DNA per well, adduct levels of approximately 1 adduct/108nucleotides can be measured.
In total up to 200 µg of DNA was required, because 3-4 wells were used per plate per sample and samples were assayed on 2 plates. In assays in which the monoadduct is isolated before analysis, sensitivity can be further increased provided larger amounts of DNA are available [1].
Dissociation-enhanced lanthanide fluoroimmunoassay
The cELISA has been modified to achieve an approximately 6-fold increase in sensitivity. This modified assay, a competitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) utilises the same antisera as the ELISA. However, the alkaline phosphatase conjugate has been replaced with a biotin-europium-labelled streptavidin signal ampli-fication system, and the release of europium into the solution forms a highly fluorescent chelate complex that is measured by time-resolved fluorometry [15]. Time-resolved fluorometry allows simple, robust assays, and above all, it results in improved sensitivity. The high sensitivity of time-resolved fluorometry stems from the unique fluorescence properties of lanthanide chelates, which allow their clear distinction from background on Biomarker identification techniques: Other techniques to detect DNA adducts
Frederik-Jan van Schooten, Roger Godschalk 32
the basis of time as well as on fluorescence wavelength. Europium chelates have a far wider Stokes shift than commonly used conventional fluorophores. This means that the wavelength of the emitted light is as much as 200–300 nm longer than that of the exci-tation light used [16]. Moreover, the fluorescence of lanthanide chelates lasts 20–35 times longer than fluorescence from a conventional fluorophore. Thus, time-resolved measure-ment starts only after fluorescence from any conventional substance would be unde-tectable, resulting in a better signal-to-noise ratio. The DELFIA method has achieved a 5- to 6-fold increase in sensitivity for measurement of DNA samples modified in vitro with benzo[a]pyrene (B[a]P), for cultured cells exposed to radiolabelled B[a]P, and for hu-man samples from occupationally exposed workers [15]. The assay has been validated by comparison of adduct levels determined by ELISA and radiolabelled genotoxins. The stan-dard curves demonstrated a limit of detection of 1.3 adducts in 108nucleotides by DELFIA,
using 20 µg of DNA per microtiter well. If 35 µg of DNA had been used in the DELFIA, the calculated detection limit would have been 0.7 adducts in 108nucleotides [15].
Chemiluminescence immunoassay
A chemiluminescence immunoassay (CIA) has been developed for several types of DNA adducts (e.g. BPDE-DNA [17] or tamoxifen-DNA [18]). Although the assay principle is simi-lar to that of a cELISA, the ultimate detection of chemiluminescence instead of a coloured end product offers much lower detection limits. Compared with DELFIA, the CIA has a lo-wer background and an approximately 10-fold increase in sensitivity (the lolo-wer limit of de-tection, using 20 µg of DNA per well, is ~1.5 adducts/109nucleotides); this sensitivity
approaches the sensitivity of 32P-postlabelling. The high signal-to-noise ratio gives a
consis-tent assay with a stability that provides a considerable advantage compared with DELFIA and previous other immunoassays [17]. The standard curves are more reproducible, largely because the problem of high background has been solved. One general disadvantage of the immunoassays, the fact that they require large quantities of DNA, has not been completely eliminated. However, when 10 µg of DNA was used per well in a BPDE-DNA CIA, only 3 out of 43 human samples gave values below the limit of detection [17]. This constitutes a substantial improvement over the earlier immunoassays that used up to 35 µg of DNA per well [17]. The fact that adduct levels in most human samples were in the detectable range for this assay suggests that the problem of performing studies where a substantial fraction of samples give a negative result because their adduct levels are below the limits of detection has been eliminated.
Immunohistochemistry
Immunohistochemical detection of DNA damage can be carried out on cells as well as tissue sections (frozen or paraffin). Several procedures can be applied to enhance anti-body binding and assay sensitivity, for example treatment with proteases to remove histone and nonhistone proteins from the DNA and/or treatment with RNase to eli-minate potential cross-reactivity with RNA adducts [1]. This can be followed by acid or base treatment to denature the DNA and further increase antibody accessibility
33 Biomarker identification techniques: Other techniques to detect DNA adducts
to the DNA adduct by antigen retrieval [19]. There are two commonly used detection systems for visualisation of adduct-bound antibodies: immunofluorescence or im-munoperoxidase. Cells can be counterstained to allow visualisation of nuclei in adduct-negative cells. Assessment of DNA adduct levels can be performed in a qualitative or semi-quantitative manner. Qualitative assessment uses subjective estimation of staining intensity and/or number of positively stained cells with an arbitrary scale [20]. In quan-titative assessment the intensity of either fluorescence or absorption of the nucleus is measured using sophisticated video cameras and software [19]. There have been attempts to convert staining intensities to absolute DNA adduct levels (e.g. number of adducts per normal nucleotide) [21]. Despite these efforts, immunohistochemical assessment of DNA adducts should currently be regarded as semi-quantitative.
For immunohistochemical assays, it is important to run proper control experiments to demonstrate the specificity of cell staining. These controls should include positive as well as negative controls. Positive controls include cells in vitro exposed to the genotoxin against which the antibodies have been raised, or tissues of exposed animals. Negative controls include samples containing adducts but treated with DNase before incubation with primary adduct-specific antibody or with primary antibody that has been preabsorbed with the DNA adduct of interest [1].
The major advantages of the immunohistochemical method are its ability to detect adducts in specific cell types within a tissue and its applicability to small amounts of sample. The major disadvantage of the immunohistochemical method is cross-reacting antibodies, which will result in errors in quantitation. In general, the method is not as sensitive as other immunoassays and quantitation is still problematic.
Immuno-dot/slot-blot methods
Slot- or dot-blot methods have also been developed to detect several types of DNA adducts, for example polycyclic aromatic hydrocarbons (PAHs) [22], alkylating [23] or malondialdehyde [24] DNA adducts. In the slot/dot-blot methodology, DNA is immobilised on nitrocellulose and subsequently incubated with primary antibodies. Subsequent incubation with enzyme-labelled secondary antiserum can result in coloured or chemiluminescent end products. Slot- or dot-blot analyses for PAH-DNA showed a sensitivity of 2 adducts per 107nucleotides [22]. Malondialdehyde-DNA adducts were
detected with a limit of sensitivity of 2.5/108 using peroxidase-conjugated secondary
antisera and a chemiluminescence end point [24]. These methods have not yet been used extensively on human samples, probably because of the low level of sensitivity. Although the immuno-slot-blot method has a relatively high absolute sensitivity (attomoles), it may not reach the sensitivity required for human DNA samples, due to the limited amount of DNA that can be used in the assay (≤ 3 µg).
Fluorescence detection of DNA adducts with fluorescent properties
Some adduct types, or their hydrolysis products, have fluorescent characteristics that can be used for their sensitive detection and quantitation. Combining the fluorescent
Frederik-Jan van Schooten, Roger Godschalk 34
characteristics (specific excitation and emission wavelengths) with high performance separation techniques [e.g. high performance liquid chromatography (HPLC)] will even make it possible to detect stereoisomers. Overall, the major limitation of the use of fluo-rescence spectroscopy for the detection of carcinogen-DNA adducts is the requirement that the adduct is intrinsically fluorescent. An additional disadvantage is that to reach sufficient sensitivity large quantities of sample DNA (100–1000 µg) are necessary. An advantage is that assays can be performed rapidly and inexpensively, once the initial cost of the equipment has been incurred.
High-performance liquid chromatography with fluorescence detection
The best example of a DNA adduct detected by fluorescence is the benzo[a]pyrene (B[a]P)-DNA adduct, which is frequently detected and quantitated by using the fluorescence of its hydrolysis products, B[a]P-tetrols [25]. The fluorescence of the BPDE-DNA adduct is much lower than the fluorescent capacity of its tetrols, which are released from the DNA after acid hydrolysis. Alexandrov et al. [26] have increased the sensitivity of this assay so that it is now sufficient to detect specific BPDE-DNA adducts in human samples. Although, this assay requires relatively high amounts of DNA, it has been successfully applied to samples from humans exposed to cigarette smoke [27] or occupa-tionally exposed to PAHs [28], and coal tar-treated patients [29]. This assay, with a de-tection limit of one B[a]P adduct in 108unmodified nucleotides, was validated for
quanti-fication of B[a]P-DNA adducts in human white blood cells by 32P-postlabelling. A highly
significant correlation and proportionality was found between the B[a]P-DNA adduct levels measured by fluorescence and 32P-postlabelling (r = 0.95; p = 0.001) [30]. A
com-parative study with B[a]P-exposed rats showed a detection limit for HPLC-fluorescence detection (FD) analysis varying from 0.5 to 7.4 adducts per 108 nucleotides, while for
postlabelling this was around 1 adduct per 109nucleotides [31]. The HPLC-FD assay can
be used to identify BPDE isomers with different biological effects and might therefore be of value in the risk assessment of individuals exposed to PAHs.
Synchronous fluorescence spectrophotometry
For the measurement of BPDE-DNA adducts, a sensitive fluorescence assay was developed, based on synchronous fluorescence spectrophotometry (SFS). Synchronous fluorescence spectrophotometry was originally presented by Lloyd [32] and applied by Vähäkangas et al. [33] to detect BPDE-DNA adducts. One hundred micrograms of DNA was hydrolysed in 0.1 M HCl at 90°C for three hours. Hydrolysis products of BPDE-DNA adducts were then measured by SFS using a fluorescence spectropho-tometer. In SFS, when the excitation wavelength is 345 nm, the peak formed correlates linearly with the level of adducts when scanned synchronously with a wavelength difference of 34 nm [33]. One fluorescence unit equals approximately 1.1 fmol BPDE/g DNA. The sensitivity of SFS is about 1 adduct/107 nucleotides. Theoretically,
syn-chronous fluorescence spectroscopy can be applied to resolve multiple components in mixtures without separation. For instance, fluorescence emission maxima occur
35 Biomarker identification techniques: Other techniques to detect DNA adducts
at 379 nm for B[a]P-tetrols and -triol, which are hydrolysis products of BPDE. Similarly, the peak for pyrene is at 372 nm and for 1-nitropyrene at 386 nm. Synchronous fluorescence spectrophotometry can also be used to measure femtomole levels of aflatoxins, their metabolites and DNA adducts [34].
Fluorescent labelling of non-fluorescent DNA modifications
Capillary electrophoresis and laser induced fluorescence
An ultrasensitive assay for measuring DNA damage was described recently that couples immunochemical recognition with capillary electrophoresis and laser-induced fluorescence (CE-LIF) detection [35]. An absolute detection limit for thymine glycols at the zeptomole level was reported [35]. However, it is still questionable whether a good overall detection limit can be reached (relative adduct level), because in CE only a small volume can be injected into the system. Another approach is the chemical linkage of fluorescent dyes to DNA adducts, which can subsequently be separated and detected by CE-LIF. A labelling procedure was described by Schmitz et al. [36] that resulted in a detection limit of 2 adducts per 106nucleotides for several types of adducts (including
adducts formed by B[a]P, aristolochic acid and etheno-DNA adducts). The limit of detection could be further improved to 1.4 per 107nucleotides by using
electrostack-ing [36]. Obviously, this methodology is not sensitive enough to be applied to human samples, but it should be mentioned that its potential is not yet fully exploited and deserves further study.
Mass spectrometrically detected DNA adducts
Gas chromatography-mass spectrometry (GC-MS) is highly specific and has been widely applied for the measurement of carcinogen-protein adducts [37] and to a lesser extent for carcinogen-DNA adducts [38–40]. The major advantage of GC-MS as compared with other techniques is that it provides information on the molecular weight and structure of the DNA adduct, and thus contributes to a positive identification. Furthermore, it can help to identify unknown adducts and their structures, which may be important in explaining the mechanisms of action of the genotoxic agents under study. Though several GC-MS methods have been developed, so far applications for carcinogen-DNA adduct measurements in biological matrices have been limited, partly because of the great expense and the relatively large amount of sample needed for analysis to reach sensitivity. Combined liquid chromatography-electrospray ionization-mass spectrometry (ESI-LC-MS) has been applied to analyse DNA adducts at the nucleotide level without the prior derivatization that is needed for GC. This allows the study of sugar-phosphate modifications, which is not possible with GC-MS [41]. Subsequently, the sensitivity was improved by combining it with column-switching techniques and nanoscale liquid chromatography, which also allowed on-line sample cleanup by removing the unmodified 2’-deoxynucleotides [42]. Such a NanoFlow ESI-LC-MS system improved the mass sensitivity by a factor of 3300, and demonstrated different DNA base- and
phosphate-Frederik-Jan van Schooten, Roger Godschalk 36
alkylated adducts of bisphenol A diglycidyl ether. Further experiments are needed to prove the value of this technique in the in vivo situation.
Section 2.1.3. decribes the use of mass spectrometry for the detection of DNA adducts in more detail.
Other techniques
Other techniques to assess DNA adduct levels are available, and have been discussed in great detail in reports from previous EU projects {e.g. the European Standards Committee on Oxidative DNA Damage (ESCODD) [43]}. These assays predominantly measure oxidative DNA damage, but can be converted to measure other types of DNA damage as well, and are therefore briefly discussed in this section.
Electrochemical detection of DNA adducts
The electrochemical detector responds to any substance that is either oxidisable or reducible and the electrical output results from an electron flow caused by the chemical reaction that takes place at the surface of the electrodes. When the mobile phase of the HPLC system is flowing past the electrodes, the solute will be continuously replaced and a current will be maintained (albeit with varying magnitude). Oxidisable or re-ducible compounds that pass the detector will change this current in a quantitative manner. Examples of DNA adducts that can be detected by electrochemical detection are 8-oxo-dG and 8-nitro-dG [44,45].
The comet assay
The comet assay (single-cell gel electrophoresis assay) is a versatile assay that can be employed as a rapid tool to assess several types of DNA damage, but predominantly single- and double-strand breaks. The comet assay is increasingly used to evaluate DNA damage in vitro as well as in vivo. The alkaline comet assay (pH > 13) has high sensitivity due to the detection of a broad range of DNA-damaging events that includes covalently bound adducts and reactive oxygen species in single cells. This broad spectrum of DNA damage can be readily assessed in a short period of time using as few as 10,000 cells per sample. The comet assay has been further modified to be able to detect several types of DNA damage; the detection of 8-oxo-dG by using the formamidopyrimidine DNA glycosylase (Fpg)-comet assay is probably the most important example of such a modification (reviewed in [46]).
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