3.1. Biomarkers of DNA base oxidation
Ryszard Olinski
1and Steffen Loft
21 Nicolaus Copernicus University, Bydgoszcz, Poland 2 University of Copenhagen, Copenhagen, Denmark
3.1.1. Mutagenic and carcinogenic properties of DNA base derivatives
It has been shown that free radical attack upon DNA generates a whole series of DNA
changes, among them modified bases. Hydroxyl radical (•OH) attack on DNA leads to
modification of a large number of pyrimidine and purine bases. Some of these modified
DNA bases have considerable potential to damage the integrity of the genome [1,2].
8-Oxo-7,8-dihydroguanine (8-oxoGua) is one of the most critical lesions. The presence
of 8-oxoGua residues in DNA leads to GC to TA transversion unless repaired prior to
DNA replication [3]. Therefore, the presence of 8-oxoGua may lead to mutagenesis.
Furthermore, many observations suggest a direct correlation between 8-oxoGua
formation and carcinogenesis in vivo [4,5]. Thus, it has been found that oxyradicals induce
mutagenesis of hotspot codons of the human p53 and Ha-Ras genes [6,7]. In agreement
with this finding GC to TA transversions have been frequently detected in the p53 gene
and in the ras protooncogene in lung carcinomas and primary liver cancer [3,6,7].
(It should be mentioned, however, that GC to TA transversions are not only caused
by 8-OH-Gua). In this context it is noteworthy that elevated levels of typical free
radical-induced DNA base modification, including 8-oxoGua, have been demonstrated in
human cancerous lung tissues compared with cancer-free surrounding tissue [8,9].
The mutagenic and carcinogenic potential of any modified DNA base is reflected
in its miscoding properties. It has been demonstrated that several other bases may have
miscoding potential. Thus, the presence of 2-hydroxyadenine (2-OH-Ade) in DNA may
induce A to C and A to T transversions and A to G transitions [10]. It was also shown
that 2-hydroxy-deoxyadenosine triphosphate (2-OH-dATP) is a substrate for DNA
polymerase and may be incorrectly incorporated by this enzyme [10].
8-Hydroxy-adenosine (8-OH-Ade) also has miscoding properties and induces mutation in
mamma-lian cells [11]. 5-Hydroxycytosine (5-OH-Cyt) has been shown to be a potentially
pre-mutagenic lesion leading to GC to AT transitions and GC to CG transversions.
5-Hydroxycytosine appears to be more mutagenic than any other product of oxidative
DNA damage [5]. It is also possible that derivatives of guanine other than 8-oxoGua
have miscoding properties [12]. On the other hand the biological consequences
of other products of base modification, like 4,6-diamino-5-formamido-pyrimidine
(FapyAde), 5,6-dihydroxyuracil (5,6-diOH-Ura) and 5-hydroxy-5-methylhydantoin
(5-OH-5-MeHyd), have not been investigated. It is conceivable that these lesions may
be premutagenic as well.
A series of repair systems work in concert for defence against
8-oxo-7,8-dihydro-2’-de-oxyguanosine (8-oxodG) in DNA. Oxidised guanine in DNA is mainly repaired by
oxoguanine glycosylase (OGG1), which removes 8-oxoGua opposite cytosine in the DNA
strand [13–15]. In addition, repair of 8-oxodG may to some extent occur by nucleotide
excision repair, nucleotide incision and transcription-coupled mechanisms [16]. A
speciali-sed enzyme (MTH1 or NUDT1) ‘sanitises’ the nucleotide pool by cleaving phosphates
of 8-oxodGTP, which, if incorporated during DNA synthesis, are highly mutagenic; mice
deficient in this enzyme develop tumours [17]. MYH is a base excision repair protein
removing adenine misinserted opposite oxidised guanine and working in concert with
MTH1 [18,19].
Determination of free radical-induced damage in DNA
The major problem in measurement of oxidised bases in DNA is spurious oxidation
occurring during sample preparation; DNA extraction is a critical issue [European
Standards Committee on Oxidative DNA Damage (ESCODD) [20–24]. The comet assay
detects DNA strand breaks and abasic sites; detection of base oxidations with this assay
requires the use of repair enzymes, such as formamidopyrimidine glycosylase (Fpg)
or endonuclease III, which nick DNA at oxidised purines and pyrimidines, respectively
[25]. It is recommended for detection of these lesions with minimal risk of spurious
oxidation [23,24]. However, this type of enzyme-based assay cannot determine the exact
level of specific base oxidations. In order to understand the biological consequences of free
radical induced damage in cellular DNA, chemical characterisation and quantification
of these lesions may be important.
The most widely used assay for measurement of oxidised DNA bases is based on
enzymatic degradation of extracted DNA to nucleosides, separation by HPLC and
deter-mination of 8-oxodG and dG by electrochemical (EC) and UV detection, respectively.
There are several other methodologies that are currently used to analyse the base products
but those based on mass spectroscopic (MS) detection [with liquid chromatography
(LC)- or gas chromatography (GC)-MS] are the only techniques that can unequivocally
identify a wide spectrum of oxidatively modified DNA bases. Quantification of samples
by the GC-MS technique is achieved by adding an appropriate internal standard to the
DNA sample. Custom-synthesised, stable isotope-labelled analogues of a number of
mo-dified bases that allow the application of this very accurate procedure, often called
isotope-dilution MS, have been obtained for quantitative determination of oxidative base
damage in DNA [26].
Isotope-dilution MS has been used successfully to analyse base products in DNA
isolated from cancerous and noncancerous human tissues [27,28]. It has been reported
that spurious oxidation of DNA bases could occur particularly during sample preparation
for GC-MS analysis, mainly during the acid hydrolysis that liberates purines and
pyri-midines from DNA, and during the derivatization step [29]. To avoid formation of such
artefacts during GC-MS analyses, another approach has been proposed involving
the liberation of base products from DNA by repair endonucleases (Fpg and
endonucle-ase III), instead of acid hydrolysis. The GC-MS technique was then used to identify
oxida-tively modified purines and pyrimidine [30]. This modification gives values of 8-oxoGua
in cellular DNA quite comparable to those obtained with the HPLC-EC technique.
The amount of 8-oxoGua in human lymphocytes was found to be 1.2 per 10
6Gua with
this approach, comparable to estimates from other enzymatic assays (comet assay,
alkaline elution or alkaline unwinding) [25,31]. In fact, the current consensus
of ESCODD is that the actual level of 8-oxoGua in human lymphocytes is from 0.5
to 5 per 10
6Gua [24]. However, in enzyme-based methods, Fpg-sensitive sites were
calculated instead of true 8-oxoGua. The modified GC-MS assay was also able to detect
Fapy-Ade and -Gua (9 and 10 moieties, respectively, per 10
7bases). Pyrimidine
modifications detected by this assay in human lymphocytes comprise 5-OH-Cyt
(2 moieties per 10
7bases), and 5,6-diH-Ura (9 modifications per 10
7bases).
Environmental factors affecting biomarkers of oxidative DNA damage in white blood cells
The effects of environmental and life-style factors on biomarkers of oxidative DNA
damage have been extensively studied and an exhaustive review will not be
attemp-ted here. A number of studies have found the expecattemp-ted higher levels of 8-oxodG
and other oxidatively modified bases or strand breaks in leucocyte DNA from smokers
as compared with non-smokers, although this is far from consistent [32–35].
Inconsistencies may be due to possible induction of OGG1 activity by smoking [33,36].
Exposure to ambient air particles and benzene has consistently been associated
with high levels of 8-oxodG in lymphocytes [37–40]. Many occupational exposures
have been shown to increase the level of strand breaks in leucocytes [34]. Attempts
to reduce the level of oxidative DNA damage in leucocytes by means of
anti-oxidant supplements in well nourished people have to a large extent been negative,
whereas some studies with antioxidant-rich food show positive effects [41,42].
Exercise may also modulate oxidative DNA damage: strenuous activity, e.g. under
hypoxic conditions, can increase levels [43,44], whereas moderate daily exercise
has been shown to increase MTH1 mRNA expression and reduce levels of 8-oxodG
in leucocytes [45].
The intraindividual variation in the level of 8-oxodG/Gua, strand breaks and oxidative
base damage measured by the enzyme-based comet assay is substantial and can be
difficult to distinguish from the interindividual and and/or assay variation in repeated
measurements [37,40,46,47]. For 8-oxodG/Gua and probably other DNA base
modifications in lymphocytes, storage problems at present preclude the collection
of repeated samples over time from the same individual for reliable batch analysis
However, for the enzyme-based comet assay, frozen storage in medium is possible and
for fpg-sensitive sites the intraindividual variation was substantially reduced in a study
with 8 repeated samples from different days if the samples from the same individuals
were assayed in batches [40].
3.1.2. Urinary measurement of oxidatively modified bases and nucleosides
The background level of 8-oxoGua in cellular DNA represents a dynamic equilibrium
between the rate of formation of oxidative DNA damage, and the rate of repair in the
specific tissue/cells studied. Numerous, highly redundant, repair processes have evolved
precisely to prevent 8-oxoGua persisting in DNA, a clear indicator of the importance this
lesion has in disrupting genome stability. Normal metabolic processes can give rise to
8-oxoGua and, as a consequence, levels of this lesion can be detected in cells (so-called
background levels). However, controversy surrounds the issue of exactly how much
damage is present, not least due to the potential for artefactual damage to occur during
extraction of DNA from cells (see also above). ESCODD was formed to resolve the
problems associated with the measurement of background levels of oxidative damage to
DNA (in particular 8-oxoGua) in human cells. As a result of these endeavours, assays for
this damage have become more precise and accurate [20-24]. Instead of measuring damage
in specific cells, with concomitant problems such as artefact formation, a whole body
burden of oxidative stress may be assessed by the measurement of urinary excretion
of 8-oxoGua, and its deoxynucleoside equivalent 8-oxodG [32,48–51].
The analysis of 8-oxoGua in urine presents particular difficulties (i.e. poor solubility
can cause loss of the analyte [33]) and, until recently, there has been no reliable assay
for its detection. New techniques, based upon MS detection, have been developed
that allowed for the simultaneous determination of 8-oxodG, 8-oxoGua and
5-methylhydroxyuracil (5HMUra) in the same urine sample [52–54]. One such method
involves HPLC prepurification followed by GC with isotope-dilution MS detection [52].
In addition to unequivocal identification of the analysed compounds and high
sensitivity, the use of isotopically-labelled internal standards compensates for potential
losses of the analytes during sample work-up. Other assays used for measurement
of urinary concentrations of 8-oxodG include HPLC-EC, CE-EC, HPLC-MS/MS and
enzyme-linked immunosorbent assay (ELISA) [48]. ELISA assays have suffered from
problems of insufficient specificity of antibodies although recent developments may
have reduced this problem [55]. Other oxidative base damage products that have been
measured in urine include thymine glycol, thymidine glycol, 8-oxoadenosine,
8-oxodeoxyadenosine, as well as various repair products of DNA damage secondary
to lipid peroxidation, including etheno and malondialdehyde adducts to guanine,
cytosine and adenosine [52–54,56–61].
It is understood that, following excision from DNA, the oxidatively modified lesions
are released into the bloodstream and consequently appear in the urine. There is
a common belief that the presence of 8-oxodG in urine represents the primary repair
product of the oxidative DNA damage in vivo and that this compound may reflect the
involvement of the nucleotide excision repair pathway (NER) [32,49] and/or the activity
of MTH1 on 8-oxodGTP) [49]. Recently, an in vitro study showed that 8-oxodG
is excreted from cells after exposure to oxidative stress resulting from exposure to ionizing
radiation [62]. However, oxidatively damaged DNA bases are primarily repaired by
the base excision repair pathway (BER), although NER may also play a role in the repair
of some oxidised bases in DNA [63], particularly under certain cellular conditions [16].
Moreover, several glycosylases which specifically recognise and remove 8-oxoGua in
human cells have recently been described [64–66], whereas the route by which 8-oxodG
arises in extracellular matrices is less clear. Therefore, urinary assays that measure
8-oxoGua reflect products of glycosylase activities, and those that measure 8-oxodG
reflect products of other, as yet undefined, activities.
3.1.3. Sources of urinary lesions
For a meaningful interpretation of results derived from different assays of urinary,
oxidatively modified bases/nucleosides, there is a need to answer a key question “from
where do these lesions originate?” Data from our laboratories, supported by literature
reports, have been largely responsible for exonerating urinary 8-oxoGua and 8-oxodG
levels from the influences of diet, cell death/turnover and artefactual formation
(for review see Olinski et al. [50]). In the absence of these confounding factors, urinary
8-oxoGua and 8-oxodG may be attributed entirely to DNA repair, although the exact
contribution of the putative processes, glycosylases, NER, endonuclease(s) and MTH1/
/8-oxodGTPase activity, plus any undiscovered activities, remains to be established. The
removal of these impediments to the interpretation of urinary data presents new and
exciting challenges, not only determining the relative contributions of the repair
pathways to the levels of urinary lesions, but also investigating modulation of DNA
repair and associations between repair and disease.
It is also noteworthy that the combined amount of 8-oxoGua, 8-oxodG and 5-HMUra
excreted into urine of healthy human subjects corresponds to about 2800 oxidative
modifications of guanine per cell per day, based on mouse data [51,59,67].
Urinary excretion of 8-oxoGua and 8-oxodG in cancer patients
Since the level of modified nucleosides/bases in urine may be an indicator of oxidative
insult to DNA, a general marker of oxidative stress, or perhaps reflective of DNA
repair, the amount of 8-oxoGua and 8-oxodG in urine from cancer patients was
examined. It was found that the amount of the modified base, but not the
de-oxynucleoside, excreted into urine was approximately 50% higher in cancer patients
than in the control group [51]. The level of the lesions in urine can depend on oxidative
DNA insult. Therefore, the higher level of 8-oxoGua in urine of cancer patients may
be explained, at least partially, by the reported oxidative stress in cancer tissue [8,9,68].
However, the amount of the modified base/nucleoside excreted into urine
should represent the average rate of DNA damage in the whole body [32,49]. Therefore,
it is doubtful whether the elevated level of the base product in cancerous cells alone
could account for the observed 50% increase of 8-oxoGua in urine. Results presented
above suggest that, rather than representing increased oxidative stress in just
the tumour, cancer patients have a subtly raised level of oxidative stress in other tissues
(or the whole body).
It is also possible that a prooxidant environment is characteristic of advanced stages
of cancer and that oxidative stress is, rather, a result of disease development.
Accordingly, cohort studies are required to assess whether an increased level
of biomarkers of oxidative DNA damage is actually associated with an increased risk
of developing cancer [69].
Environmental factors affecting urinary excretion of 8-oxodG and 8-oxoGua
The urinary excretion of 8-oxodG and 8-oxoGua has relatively consistently been found to
be increased among smokers [32,33,70]. Heavy exposure to air pollution in occupational
settings in terms of, e.g. diesel exhaust, polyaromatic hydrocarbons and benzene has been
associated with increased 8-oxodG excretion [70–72], whereas non-occupational exposure
to ambient air pollution was not associated with 8-oxodG excretion [37,73]. Exercise
of very high intensity, e.g. marathon running, or at high altitude has been associated with
increased 8-oxodG excretion [43,74,75]. Cancer treatment with radiation and/or
chemotherapy may also increase 8-oxodG excretion [77,78]. A number of studies
have investigated the potential effects of diet and antioxidants including
interven-tions on urinary 8-oxodG excretion, showing generally no effect [47,78]. Importantly,
the urinary excretion of 8-oxodG is relatively constant within an individual,
i.e. intraindividual variation is 20% or less in studies with repeated measurements.
Moreover, 8-oxodG is extremely stable in urine samples and the levels are unchanged
in samples stored for 15 years at –20°C.
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3.2. Lipid peroxidation induced DNA damage
Urmila Nair and Jagadeesan Nair
German Cancer Research Center, Heidelberg, Germany
Chronic inflammatory processes produce an excess of reactive oxygen (ROS) and reactive
nitrogen species (RNS) and DNA-reactive aldehydes from lipid peroxidation (LPO). The
persistent oxidative stress and excess lipid peroxidation (LPO) that are induced by
inflam-matory processes, impaired metal transport or dietary imbalance cause accumulation
of massive DNA damage together with deregulation of cell homeostasis [1]. These events
play an important role in human chronic disease pathogenesis. DNA damage caused by
ROS, RNS and LPO endproducts provides promising markers for risk prediction and targets
for preventive measures. Among these, 4-hydroxy-2-nonenal (HNE), malonaldehyde
(MDA), acrolein, and crotonaldehyde have been the most widely studied aldehydes with
respect to their chemical and biological interactions with nucleic acid bases. The ability
of these reactive electrophiles to alkylate DNA bases, yielding the respective promutagenic
lesions, has been considered to contribute to the mutagenic and carcinogenic effects
associated with the oxidative stress-induced lipid peroxidation process.
Lipid peroxidation products generate exocyclic adducts with either a five-member ring
(etheno adducts) or a six-member ring (propano adducts). The structures of the main
adducts measured in human studies are depicted in Fig. 3.1. The etheno adducts include
1,N
6-etheno-2’-deoxyadenosine (
εdA), 3,N
4-etheno-2’-deoxycytidine (
εdC), 1,N
2-etheno--2’-deoxyguanosine (1,N
2εdG) and N
2,3-etheno-2’-deoxyguanosine (N
2,3
εdG). The
propa-no adducts are 1,N
2-propano-2’-deoxyguanosines designated AdG and CdG representing
the aldehydes responsible for their generation namely acrolein and crotonaldehyde and
pyrimido[1,2-
α]purine-10(3H)-one-2’-deoxyribose (M1dG) (reviewed by [1–6]).
Endogenous lipid peroxidation products such as MDA, crotonaldehyde and HNE
are particularly potent in forming adducts during periods of oxidative stress. The type and
quantity of fatty acids in the diet is also of significance in that trans-fatty acids and their
metabolic derivatives seem to lead to excessive formation of adducts.
Modified DNA base adducts that are generated by reactions of DNA with two major
LPO endproducts, HNE to yield etheno-adducts, and MDA to form MDA-derived DNA
adducts such as M1dG, have been quantified in human tissues and body fluids. The
DNA-reactive LPO products HNE and MDA are increasingly implicated in the carcinogenesis
process [51]. These intermediates can react with DNA bases to form exocyclic DNA
adducts of which several have been characterized as propano- and etheno-DNA-base
adducts [52,53]. Of the latter,
εdA, εdC and N
2,3-
εdG have been detected in vivo.
Etheno-DNA adducts appear to be important as risk markers in human disease. These
promutagenic, chemically stable markers appear to be useful for assessing oxidative
stress-derived DNA damage. The biological significance of etheno-DNA adducts is established.
Vinyl chloride is a human carcinogen (IARC Group 1) and urethane is a multi-organ
rodent carcinogen classified as a possible human carcinogen (IARC Group 2b).
AdG, acrolein-dG; CdG, crotonaldehyde-dG; HNE, 4-hydroxy-2-nonenal; M1dG, pyrimido[1,2-α]purine-10(3H)-one-2’-deoxyribose; MDA, malonaldehyde; N23εdG, N2,3-etheno-2’-deoxyguanosine
Liver Immunoaffin-32P εdA 0–2.6/108dA 10 Normal, non-tumour, cirrhotic, [10] εdA, εdC εdC 0–2.7/108dC sudden infant death livers 4–5/1010parent nt,
25–50 µg DNA
WBC, healthy Immunoaffin-32P M, MUFA, PUFA; F, MUFA 22 M and F on MUFA or PUFA [11] volunteers εdA, εdC; [10] εdA 0.3–25.2/108dA diets
εdC 0.4–11/108dC F, PUFA
εdA 1.3–902/108dC εdC 0.6–716/108dC
Liver Immunoaffin-32P C εdA 1.24–2.82/108 dA 26 Control, WD, PHC [12] εdA, εdC; [10] εdC 0.98–3.8/108dC
WD ∼3×C; PHC ∼2.3×C
Pancreas Immunoaffin-32P εdA 0.5–4/108normal nt 30 S and NS, no difference [13] εdA, εdC; [10] εdC 0–2.5/108normal nt
Colon Immunoaffin-32P εdA C 1.4–7.1/108dA 31 Control, FAP and CRC [14] εdA, εdC; [10] FAP 0.4–14.5/108dA
εdC C 0.6–2.7/108dC FAP 0.5–6.6/108dC
Leucocytes Immunoaffin-32P εdA 0–16.9/108dA 42 Questionnaire-based high [15] WBC εdA, εdC; [10] εdC 2.6–67.1/108dC and low fatty acid diets.
Lung Immunoaffin-32P εdA 0.26–14.6/108dA 23 Tumour adjacent normal tissue [16] εdA, εdC; [10] εdC 0.16–39.6/108dC smokers and non-smokers
Liver LC-ES-MS/MS εdA 0.07–0.17/108nt 4 [17]
modi-εdA, εdC εdC 0.05–0.1/108nt fied from
< 1 adduct/108nt [18]
Lung Immunoaffin-32P εdA 0.34–6.15/108A 33 Normal lung, tumour and [19] leucocytes εdA, εdC; [10] εdC 0.67–9.19/108C leucocytes from lung cancer
cases
Liver, lung, Immunoaffin-32P εdA 0.4–11/108A 12× ∼7 Diverse cancer and non-cancer [20] kidney, colon, εdA, εdC; [10] εdC 0.91–15/108C autopsy sample; cerebrum
cerebellum, highest
cerebrum
Liver IHC Quantitated by % number Control n = 15 [21]
of positive stained nuclei/ Disease n = 39 total number of nuclei
Pancreas, Immunoaffin-32P CPεdA 6.3±5.6/108dA 28, 20 Pancreas NP, 28; CP, 20 [22] colon εdA, εdC; [10] εdC 35.4±25.1/108dC Colon N, 18, CD, 18;
NPεdA 1.9±0.1/108dA UC, 13; IBD, 5 εdC 1.1±0.9/108dC N εdA 2.8±2.3/108dA εdC 1.7±1.3/108dC CDεdA 4.1±4.0/108dA εdC 32.5±34.6/108dC UCεdA 1.2±1.0/108dA εdC 6.9±3.6/108dC
Table 3.1. Exocyclic DNA adducts in tissues (human studies)
Table 3.1. Exocyclic DNA adducts in tissues (human studies) – cont.
DNA source Method/sensitivity Range n Comments Reference
Liver High resolution 56–713 fmol/µmol Gua 12 Tissue repository [6]
modi-GC-MS fied [23]
N2 ,3-etheno-guanine-εG
Liver HPLC/32P/TLC/ 30–200 adducts/108Gua 5 Autopsy samples (actual [24] /HPLC (OH-PdG) Combined range for rats, values not given)
Acrolein-dG (AdG), n = 8, and human, n = 5, Crotonaldehyde-dG samples
(CdG)
Leucocytes, AdG and CdG AdG+CdG 3 mammary [25]
breast 0.01–0.80 µmol/molG 3 leucocyte
Gingival 32P-HPLC AdG NS 9.2/108nt 23 Smoker and non-smokers [26] tissue Acrolein-dG (AdG), S 27/108nt Range, 0–3.6 µmol/mol G
Crotonaldehyde-dG1, CdG1 NS 1.2/108 nt Crotonaldehyde-dG2 S 11/108nt
CdG2 NS 6.2/108nt S 34/108nt
Liver, colon HPLC/32P/TLC/ 0.06–0.18/108nt 5 Actual values not given [8] /HPLC Combined range for rats, Liver = 3, colon = 2
HNE-dG n = 8, and human, n = 5, 1N2propanodG samples
Colon, brain 32P Colon 37.8±0.5/108nt [27]
HNE-dGp Brain 18.5±0.6/108nt
Colon (recto- 32P Mean adducts 8× Controls on low and high [28]
sigmoidal HNE-dGp; [27] Low RS 26.9±3.9/108nt resistance starch diet
biopsy) High RS 38.3±6/108nt
Brain 32P ∼2–60 adducts/108nt C, 10; Postmortem [29]
HNE-dGp; [27] C vs AD, NS AD, 13 Hippocampus > parietal cortex or cerebellum Liver GC-MS 50–120 6 [30] M1dG TWBC, breast 32P/RP-HPLC WBC 10–50; 26 [31] M1dG Breast 7–56 7 10 µg DNA
Breast 32P Cases 0.5–13/108nt 51 Normal tissue breast cancer [32] M1dG Controls 0.2–1.9/108nt 28 and non-cancer women
Leucocytes 32P MUFA 2–25/108nt 13 Females on high MUFA, [33]
M1dG PUFA 12–280/108nt PUFA diet
Leucocyte GC-MS, 5–8 adducts/108 10 [34] Immunoaffin-GC-EC, NCI-MS M1dG 100 fmol/sample — 3 adducts/108nt for 1 mg DNA 10 [34]
Table 3.1. Exocyclic DNA adducts in tissues (human studies) – cont.
DNA source Method/sensitivity Range n Comments Reference
Pancreas GC-EC NCI-MS 1–50/108nt [13]
M1dG
Pancreas 32P/HPLC 36 adducts/108nt [35]
M1dG
Liver, Lung 32P-HPLC 14 adducts/108nt 10 [35]
M1dG 10 adducts/108nt 10 µg DNA WBC ISB 5.6–9.5/108nt WBC 8 [36] Gastric M1dG 3.1–64.3/108nt Gastric 42 mucosa 2.5 adducts/108nt; 1 µg DNA
WBC ISB 7–166.5/108nt Leucocytes 5 controls on special diet [37]
M1dG; [36] = 5× (28 samples at time interval
samples?)
Gastric (antral ISB 56–60 adducts/108nt 124 Gastritis with or without [38]
mucosa) M1dG H. pylori
Colorectal ISB 0–122/108 13 [39]
biopsy M1dG; [36]
Liver LC-MS/MS Not detected 4 [17]
M1dG 1 adduct/108; 100 µg DNA
Oral mucosal IHC S, 97±41 MRSI 50 Smokers and non smokers [40]
cells MDA-DNA adduct NS, 74±17 MRSI
Liver, breast, IEP-HPLC Liver 5.2 Liver, 2 [41]
TWBC M1dG Breast 1.7 Breast, 4
200 amol from TWBC 9.5 TWBC, 26
10 µg DNA (0.6 adducts/108)
Liver ISB 25±15/108nt to Values Pre-, post-surgical samples [42]
M1G; [36] 63±52/108nt highest from curcumin treatment treatment groups
given (n, 4); rest n, 8 similar
32P, 32P-postlabelling; εC, 3,N4-ethenocytosine; εdA, 1,N6-etheno-2’-deoxyadenosine; εdC, 3,N4-etheno-2’-deoxycytidine; AD, Alzheimer disease; AdG, Acrolein-dG; C, Control;
CD, Crohn’s disease; CdG, crotonaldehyde-dG; CP, chronic pancreatitis; CRC, colorectal cancer; EC, electrochemical detection; ESI, electrospray ionization; F, female; FAP, familial adenomatous polyposis; GC-MS, gas chromatography-mass spectrometry; HNE, 4-hydroxy-2-nonenal; HPLC, high performance liquid chromatography;
IBD, irritable bowel disease; IEP, immuno-enriched 32P-postlabelling; IHC, immunohistochemistry; Immunoaffin, immunoaffinity purification; ISB, immuno-slotblot; LC, liquid chromatography; M, male; M1dG, pyrimido[1,2-α]purine-10(3H)-one-2’-deoxyribose; M1G, pyrimido[1,2-α]purin-10(3H)-one; MDA, malondialdehyde; MRSI, median relative staining intensity;
MUFA, monounsaturated fatty acid; N, normal uninvolved tissue; N2,3’dG,N2,3-etheno-2’-deoxyguanosine; NCI, negative chemical ionization; NP, normal pancreas; NS, non smoker; nt, nucleotide;
OH-PdG, 8-hydroxy-6-methyl-1,N2-propano-2_-deoxyguanosine; PHC, primary hematochromatosis; PUFA, polyunsaturated fatty acid; S, smoker; SID, sudden infant death;
Table 3.2. Urinalyses of exocylic nucleosides (human studies)
DNA source Method/sensitivity Range n Comments Reference
εC, 3,N4-ethenocytosine; εdA, 1,N6-etheno-2’-deoxyadenosine; εdC, 3,N4-etheno-2’-deoxycytidine; APCI, atmospheric pressure chemical ionization; C, control; ESI, electrospray ionization; F, female; GC-MS, gas chromatography-mass spectrometry; HPLC, high performance liquid chromatography; IP, immunoprecipitation; LC, liquid chromatography; M, male;
M1G, pyrimido[1,2-α]purin-10(3H)-one; NICI negative ion chemical ionization; NS, non smoker; S, smoker.
Urine IP-HPLC εdA 0.27–4.4 fmol/ 18 M, n = 9 [5]
εdA /µmol creatinine F, n = 9
Sensitivity 6 fmol/ /injection
Urine IP-HPLC εdA 1.2–17 fmol/ C, 30×2 Female C and Intervention (I), [43]
εdA; [5] /µmol creatinine I, 30×2 εdA
C vs I reduced
Urine GC-NICI-MS 101±17 pg εC/ml/ 2 S [44]
εC; 7.4 fmol /g/creatinine or 0.6 nmol/l
Urine GC-NICI-MS εC S, 2.65±4.0 and 23 S, NS [45]
εC; [44] NS, 0.61±0.9 ng/kg/ /gcreatinine
Urine LC-ESI-MS/MS 18 [46]
1,N6-ethenoadenine
Urine GC-NICI-MS εdC, 0–0.80 nM/24 Associated with smoking [47]
εdC modified; [45] hr urine (same samples as above?)
Urine HPLC-ESI-MS/MS; > 0.3–8 nmol/l 13 Healthy subjects (actual [48]
GC-MS values not given)
1,N2-ethenoguanine, N2,3-ethenoguanine
Urine Thermospray LC-MS Below limit 6 [49]
M1G < 500 fmol/ml
Sensitivity 500 fmol/ml urine
Urine LC-APCI-MS/MS 12±3.8 fmol/kg 5 NS, C [50]
M1G per 24 h
Hepatic etheno-DNA adduct levels were significantly elevated in patients with Wilson's
disease and primary hemochromatosis who have higher risk of developing liver cancer.
Excess storage of copper/iron causing oxidative stress and LPO-derived DNA damage are
implicated in disease pathogenesis as confirmed by studies in LEC rats, a model
for Wilson's disease. When livers of patients with alcohol-related hepatitis, fatty liver,
fibrosis and cirrhosis were compared with asymptomatic livers, excess hepatic DNA
damage was seen in all patients, except those with hepatitis. Etheno-deoxyadenosine
excreted in urine was measured in HBV-infected patients diagnosed with chronic
hepatitis, cirrhosis and hepatocellular carcinoma: compared with controls, patients
had 20–90-fold increased urinary levels (reviewed by [1]).
To facilitate dosimetry studies in humans and to provide useful biomarkers in cancer
risk assessment and biomonitoring studies, specific and sensitive methods for the
detection of these potential mutagenic adducts in human tissues and in urine have been
developed. The principal techniques developed for adduct analysis include
immuno-affinity-
32P-postlabelling, immunohistochemistry, high performance liquid
chromato-graphy with electrochemical detection (HPLC-EC), gas chromatochromato-graphy-mass
spectro-metry (GC-MS), liquid chromatography-tandem mass spectrospectro-metry (LS-ES-MS/MS),
immuno-slotblot assay and immunohistochemistry (reviewed by [4,6–9]). Tables 3.1.
and 3.2. list the important applications of these methods in human biomonitoring
studies.
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