CEBP Susan G. Komen for the Cure-AACR Outstanding Investigator Award for Breast Cancer Research Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Barbin, A.
Right arrow Articles by Swenberg, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barbin, A.
Right arrow Articles by Swenberg, J. A.
Cancer Epidemiology Biomarkers & Prevention Vol. 12, 1241-1247, November 2003
© 2003 American Association for Cancer Research

Endogenous Deoxyribonucleic Acid (DNA) Damage in Human Tissues

A Comparison of Ethenobases with Aldehydic DNA Lesions

Alain Barbin1, Hiroko Ohgaki1, Jun Nakamura2, Michael Kurrer3, Paul Kleihues1 and James A. Swenberg2

1 International Agency for Research on Cancer, Lyon, France; 2 Department of Environmental Sciences and Engineering, The University of North Carolina, Chapel Hill, North Carolina; and 3 University Hospital, Zurich, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two types of endogenous DNA lesions, ethenobases [1,N6-ethenoadenine ({epsilon}A), 3,N4-ethenocytosine ({epsilon}C)] and aldehydic DNA lesions (ADLs), were measured in several tissues (liver, lung, kidney, colon, colon mucosa, cerebellum, and gray and white matter of the cerebrum) obtained postmortem during autopsy examinations of 12 individuals (6 males, 6 females; ages, 58–87 years). Issues relating to changes in levels of DNA damage with disease and after death were addressed. The extent of DNA damage in autopsy samples was not associated with the length of the postmortem interval and was similar to levels observed in surgery samples, suggesting that endogenous, steady-state levels of etheno adducts and of ADLs are relatively stable during the hours immediately after death. In this limited series of samples, and with a few possible exceptions, the disease status before death was not associated with increased endogenous DNA damage in the affected tissue. DNA ethenobases were lowest in the cerebellum (median molar ratios: {epsilon}A/A = 1.0 x 10-8; {epsilon}C/C = 1.9 x 10-8) and highest in the gray matter ({epsilon}A/A = 2.9 x 10-8; {epsilon}C/C = 4.8 x 10-8) and white matter ({epsilon}A/A = 2.4 x 10-8; {epsilon}C/C = 5.2 x 10-8) of the cerebrum. In other tissues, median values were 1.2–1.9 x 10-8 for {epsilon}A/A and 2.0–3.3 x 10-8 for {epsilon}C/C. There was a good correlation between the levels of {epsilon}A and {epsilon}C (r = 0.80, P < 0.0001). Levels of ADLs were similar in the liver, lung, kidney, and white matter of the cerebrum (median values: 5.7–7.9 ADLs/106 nucleotides), higher in the colon (11.3 x 10-6) and gray matter of the cerebrum (9.0 x 10-6) and lower in the cerebellum (3.7 x 10-6). There was no correlation between levels of ethenobases and amounts of ADLs ({epsilon}A versus ADLs: r = 0.12, P = 0.33; {epsilon}C versus ADLs: r = 0.024, P = 0.85). Although there was an interindividual variability in the extent of endogenous DNA damage (4-fold for {epsilon}A and {epsilon}C, 2-fold for ADLs), which may be determined, in part, by the capacity to repair DNA and may be related to the pathology or treatment of the patients, these results suggest that the cerebrum contains higher endogenous DNA damage than the other tissues. These data are in line with previous studies showing that brain tissues are more susceptible to oxidative stress and lipid peroxidation than other tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The possible role of endogenous DNA damage in human diseases (degenerative diseases, cancer) is receiving increasing attention (1 , 2) . Several lesions that can lead to mutations have been characterized, the most common being oxidized bases (3) and abasic sites (4) . More recently, exocyclic DNA adducts derived from LPO4 products were detected in DNA from humans and rodents (5 , 6) . Enals such as HNE derived from PUFAs yield propanobases with DNA; if further oxidized into epoxy-aldehydes, such as 4-hydroxy-2,3-epoxynonanal, they yield ethenobases (7) . Another major DNA lesion formed by LPO products is M1G, a pyrimidopurinone adduct of malondialdehyde with guanine (8) . These exocyclic DNA adducts exhibit promutagenic properties (8, 9, 10) and, thus, could be involved in genomic instability and carcinogenesis. Several known cancer risk factors increase the level of these DNA lesions in target organs. Elevated levels of {epsilon}A and {epsilon}C have been found in hepatic DNA from patients with metal storage diseases (Wilson’s disease, primary hemochromatosis; Ref. 11 ) and in colonic polyps of familial adenomatous polyposis patients (12) . A diet rich in {omega}-6 PUFAs has been shown to increase etheno DNA adducts in WBCs of female subjects (13) . In a mouse model, overproduction of nitric oxide induced high levels of etheno adducts in spleen DNA (14) . Fulminant hepatitis in Long Evans Cinnamon rats, a strain with abnormal copper metabolism, coincided with a peak in the number of etheno adducts in liver DNA (15) . Administration of ethanol to rats enhanced the formation of endogenous etheno adducts in hepatic DNA (16) . Altogether, these data suggest that etheno DNA adducts could be useful biomarkers of DNA damage associated with oxidative stress and LPO (17) .

AP or abasic sites are the most common DNA lesions. It has been estimated that ~10,000 AP sites arise every day in the DNA from a mammalian cell (18 , 19) . These sites can result from spontaneous depurination of oxidized bases (20) , activity of DNA polymerases on oxidized or alkylated bases (20) , or from attack of deoxyribose moieties by hydroxyl radicals (21) . If not repaired, AP sites can be lethal or mutagenic (4) . However, because of a lack of a reliable and sensitive assay, only a few studies have been performed on AP sites in vivo. Recently, a sensitive slot-blot assay was developed using ARP that can react with the aldehydic forms of AP sites caused by DNA glycosylases or spontaneous depurination/depyrimidination, resulting in the formation of stable biotin-tagged complexes (19) . The adduction of aldehydic AP sites using ARP enabled us to quantitate the total number of regular AP sites by the ARP-slot blot assay. However, ARP is not a specific probe for AP sites, because it can also react with aldehydic bases and probably aldehydic oxidized deoxyribose lesions (22) . In this study, the lesions detected by the ARP-slot blot assay in human tissue DNA will be referred to as ADLs, instead of AP sites, unless there is clear evidence that indicates that they are real AP sites. Furthermore, the DNA isolation step was improved to avoid artifactual formation or degradation of ADLs (23) .

There have been few systematic studies of endogenous levels of DNA damage in different tissues from humans. To improve our knowledge on the extent of background DNA damage in humans, tissue samples were collected at autopsy, a few hours after death. These samples were analyzed for two ethenobases, {epsilon}A and {epsilon}C, and for ADLs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue Samples.
Human tissue samples were obtained from the University Hospital of Zurich, Switzerland. The tissues (liver, lung, kidney, colon, colon mucosa, gray and white matter of the cerebrum, and cerebellum) were collected through a medical examiner’s office, snap frozen in liquid nitrogen, and kept at -80°C. Gender, age, and cause of death were determined by the medical examiner. This study was approved by the IARC Ethical Review Committee.

Clinical data of the patients are shown in Table 1Citation . There were six women and six men, their ages ranged from 58 to 87 years. The postmortem interval (time at which the tissue samples were collected and frozen) ranged from 3 to 17.5 h (median ~ 8 h).


View this table:
[in this window]
[in a new window]
  		Table 1 Clinical data on the patients

 
Isolation of DNA.
For analysis of etheno adducts, DNA was isolated using the phenol/chloroform extraction method (24) . For analysis of ADLs, a modified phenol/chloroform extraction method was used as described elsewhere (23) . Briefly, to prevent Fenton reactions and oxidation of DNA, 2,2,6,6-tetramethylpiperidine-N-oxyl was added to the buffers; digestion with proteinase K was carried out at a low temperature (4°C) to prevent degradation of ADLs.

Analysis of {epsilon}A and {epsilon}C.
Fifty micrograms of DNA were hydrolyzed enzymatically into nucleoside 3'-monophosphates. 3'-{epsilon}dAMP and 3'-{epsilon}dCMP were purified on specific immunogels and postlabeled on the 5'-position with [{gamma}-32P]ATP and T4 polynucleotide kinase (6) . The labeled nucleoside 5'-monophosphates were separated by two-dimensional thin-layer chromatography on polyethyleneimine cellulose and quantitated with a PhosphorImager (Molecular Dynamics). Normal nucleotides in DNA hydrolysates were quantitated by UV detection, after reverse-phase HPLC separation. Levels of etheno adducts were expressed as molar ratios of ethenonucleotide to parent nucleotide in DNA.

Analysis of ADLs.
The ARP-slot-blot assay was performed as described by Nakamura et al. (23) . Eight micrograms of DNA in 30 µl of PBS were incubated with 1 mM aldehyde reactive probe at 37°C for 10 min. After precipitation using cold ethanol, DNA was resuspended in TE buffer [10 mM Tris-HCl (pH 7.4), containing 1 mM EDTA]. The DNA concentration was measured by a UV spectrophotometer, and the DNA solution was then prepared at 0.275 µg/220 µl of TE buffer. Heat-denatured DNA was immobilized on a nitrocellulose membrane (Protran BA85, Schleicher & Schuell). The nitrocellulose membrane was soaked with 5x SSC and then baked in a vacuum oven for 30 min. The membrane was preincubated with 10 ml of hybrid mix for 15 min and then incubated in the same solution containing streptavidin-conjugated horseradish peroxidase at room temperature for 45 min. After rinsing the nitrocellulose membrane, the enzymatic activity on the membrane was visualized by enhanced chemiluminescence reagents. The nitrocellulose filter was then exposed to X-ray film, and the developed film was analyzed using an Ultrascan XL scanning densitometer. The number of ADLs per 106 nucleotides was calculated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Etheno Adducts.
Levels of {epsilon}A and {epsilon}C were analyzed in DNA from liver, lung, kidney, cerebellum, and cerebrum from 10 individuals (a–j, Table 1Citation ), and in colon tissue and colon mucosa from 8 individuals (c–j, Table 1Citation ). Data were grouped by tissue (Figs. 1ACitation and 2ACitation ) or by individual (Figs. 1BCitation and 2BCitation ).



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 1. Levels of {epsilon}A in human DNA (autopsy tissue samples from 10 individuals identified by letters a–j). Each value is the average of two or three analyses. Horizontal bars represent medians.

 


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 2. Levels of {epsilon}C in human DNA (autopsy tissue samples from 10 individuals identified by letters a–j). Each value is the average of two or three analyses. Horizontal barsrepresent medians.

 
Levels of etheno adducts differed significantly between the eight tissues analyzed: P = 0.0015 for {epsilon}A, P = 0.0203 for {epsilon}C (Kruskal-Wallis test). The cerebellum exhibited lower levels of {epsilon}A than the gray matter and white matter of the cerebrum (P < 0.01, Dunn’s test) and lower levels of {epsilon}C than the gray matter of the cerebrum (P < 0.05, Dunn’s test). In most cases, ranges and median values of both etheno adducts differed only marginally among the liver, lung, kidney, colon, and colon mucosa. {epsilon}A/A levels ranged from 0.83 to 4.08 x 10-8 in these tissues, with median values between 1.20 and 1.86 x 10-8 (Fig. 1A)Citation . The molar ratio of {epsilon}C/C varied from 0.97 to 8.88 x 10-8, with median values of 2.05 to 3.34 x 10-8 (Fig. 2A)Citation . Two exceptions were one liver sample (patient b) and one lung sample (patient a), which contained high levels of both {epsilon}A ({epsilon}A/A = 8.27 and 12.2 x 10-8, respectively) and {epsilon}C ({epsilon}C/C = 15.3 and 16.7 x 10-8, respectively). The cerebellum exhibited the lowest levels of {epsilon}A ({epsilon}A/A, from 0.40 to 1.65 x 10-8, median, 1.00 x 10-8) and {epsilon}C ({epsilon}C/C, from 0.91 to 6.81 x 10-8; median, 1.92 x 10-8). Levels of etheno adducts were found to be higher in gray and white matter of the cerebrum: {epsilon}A/A ranged from 1.0 to 11 x 10-8 (median, 2.9 x 10-8) in the gray matter, and from 1.1 to 8.6 x 10-8 (median, 2.4 x 10-8) in the white matter; {epsilon}C/C ranged from 2.3 to 14 x 10-8 (median, 4.8 x 10-8) in the gray matter, and from 1.4 to 15 x 10-8 (median, 5.2 x 10-8) in the white matter.

When these data were grouped by individual (i.e., grouping the levels of etheno adducts measured in the six or eight different tissues for each individual), interindividual differences in the range and average levels of etheno adducts were obvious (Figs. 1BCitation and 2BCitation ): P = 0.0204 for comparison of {epsilon}A levels and P = 0.0012 for comparison of {epsilon}C levels (Kruskal-Wallis test). In addition, Dunn’s multiple comparisons test showed a significant difference between patient a, on the one hand, and patients j and g on the other hand, with regard to the levels of {epsilon}C (P < 0.05). Patients a and b and, to a lesser extent, patients c and d, exhibited a larger range and higher median values of {epsilon}A/A (Fig. 1B)Citation and {epsilon}C/C (Fig. 2B)Citation in their DNA than did patients e to j.

Comparison of the levels of {epsilon}A and {epsilon}C measured in each DNA sample showed a significant linear correlation: r = 0.80, P < 0.0001 (n = 76; Fig. 3Citation ). The slope of the correlation indicated that the average ratio of {epsilon}A/A to {epsilon}C/C in human DNA was 0.49 ± 0.04.



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 3. Correlation between levels of {epsilon}A and of {epsilon}C in human DNA.

 
ADLs.
ADLs were measured in liver, lung, kidney, cerebellum, gray and white matter of the cerebrum from 12 individuals (a–l, Table 1Citation ; except for white matter of the cerebrum from patient b and liver from patient k, which were not analyzed for ADLs) and in colon tissue from 8 individuals (c–j). This included the same tissue samples that were analyzed for etheno adducts, as well as tissues from two other individuals (k and l). Data are shown in Fig. 4, A and BCitation . Statistical analysis indicated that the levels of ADLs differed significantly among the six tissues examined: P = 0.0019 (Kruskal-Wallis test; Fig. 4ACitation ). The number of ADLs in the cerebellum was significantly different from those in the gray matter of the cerebrum and colon (P < 0.05 and P < 0.01, respectively; Dunn’s test). The colon exhibited the highest number of ADLs (from 6.0 to 42 ADLs per 106 nucleotides; median value, 11.35), followed by the gray matter of the cerebrum (4.0 to 19 ADLs per 106 nucleotides; median value, 9.05). The number of ADLs in the liver, lung, kidney, and white matter of the cerebrum were similar, ranging from 3.1 to 17 ADLs per 106 nucleotides (median values from 5.75 to 7.90 ADLs per 106 nucleotides). The cerebellum had the lowest number: 2.80 to 11.3 ADLs per 106 nucleotides (median value, 3.70).



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 4. Levels of ADLs in human DNA (autopsy tissue samples from 12 individuals identified by letters a–l). Each value is the average of three to six analyses. Horizontal bars represent medians.

 
When grouped by individual, the number of ADLs showed a 2-fold interindividual variability that was not statistically significant (P = 0.56, Kruskal-Wallis test; Fig. 4BCitation ). The median value for each patients ranged from 5.75 to 7.90 ADLs per 106 nucleotides.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here, for the first time, a comparison of two types of endogenous DNA damage in different human tissues and in different individuals. Etheno adducts have been measured in surgery samples from liver (11) , colon (12) , and lung (25) . These data are summarized in Fig. 5Citation , together with our present data on autopsy samples. For the sake of clarity, only the minimal, median and maximal values have been represented for each data set. Although there are some differences in the maximal values, the median values are very similar, when comparing surgery and autopsy samples. This suggests that endogenous, steady-state levels of etheno adducts, at least in the liver, colon, and lung, are relatively stable during the hours immediately after death. Analysis of thiobarbituric acid reactive substances in human brain (biopsy and autopsy samples) revealed that basal LPO was not significantly affected by the postmortem delay (at least up to 24 h), at room and cold-room temperature (26) . This suggests that etheno adduct levels should not change dramatically in brain DNA up to 24 h after death. Concerning ADLs, comparable data are available for the liver only. In autopsy liver samples, Nakamura and Swenberg (27) reported 8.2 ± 1.4 ADLs per 106 nucleotides (mean ± SD, n = 4). This is very close to the amount found in the autopsy samples, i.e., 6.4 ± 2.5 ADLs per 106 nucleotides (n = 11).



View larger version (31K):
[in this window]
[in a new window]
  		Fig. 5. Comparison of the levels of DNA etheno adducts measured in surgery samples (11, 12, 25; unpublished data) and autopsy samples (this study). The range (minimal and maximal values) and the median value are indicated.

 
In this study, we observed similar amounts of endogenous DNA damage in different tissues, with some exceptions. The cerebellum contained less DNA damage (both etheno adducts and ADLs) than the other tissues examined. The levels of the etheno adducts, {epsilon}A and {epsilon}C, were higher in the gray and white matter of the cerebrum in approximately half of the samples. Higher levels of {epsilon}A and {epsilon}C have been observed previously in rat brain than in other tissues, including liver, lung, and kidney (28) . ADLs were more frequent in colon tissue, which is consistent with a previous study showing higher levels of ADLs in colon DNA than in liver, lung, and kidney DNA of rats (27) . In the same study, high levels of ADLs were found in whole brain DNA of rats; however, more recent studies using the same procedures that were used in this study did not find any difference between brain and other tissues.5 In human cerebrum, the gray matter, but not the white matter, contains slightly higher levels of ADLs (Fig. 4A)Citation .

The brain is highly sensitive to oxidative imbalance as a result of its high oxygen consumption rate, high content of transition metals and ascorbate (which together act as potent prooxidants), and the relative paucity of antioxidant enzymes compared with other tissues (29) . Brain tissue is more lipid-rich than most other tissues and contains high levels of PUFAs, such as docosahexaenoic acid and arachidonic acid, that are easily peroxidized (30) . Arachidonic acid, an {omega}-6 PUFA, is thought to be the major source of HNE and of HNE-DNA adducts in mammalian cells (31 , 32) . Consequently, the brain is more susceptible to oxidative damage and LPO than other organs (33) . A study on rat tissues has shown higher levels of aldehydes (thiobarbituric acid reactive substances) in brain as compared with the other tissues, including liver, lung, kidney (34) . Our present data showing higher amounts of {epsilon}A and {epsilon}C in gray and white matter of the cerebrum further suggest that the rate of LPO is relatively high in the cerebrum. In contrast, the cerebellum exhibited lower levels of etheno adducts. This is in keeping with studies in rats showing that the gray matter of the cerebrum is more susceptible to lipid peroxide formation than the cerebellum (35 , 36) and that background levels of malondialdehyde are higher in gray matter of the cerebrum than the cerebellum (37) .

Comparison of the median values (obtained from six to eight different tissues from each individual) shows an interindividual variability of 4-fold for the amounts of {epsilon}A (Fig. 1B)Citation and {epsilon}C (Fig. 2B)Citation and of 2-fold for the level of ADLs (Fig. 4B)Citation . This may reflect interindividual variations in DNA repair activities, which were not determined in the present study. Endogenous levels of these etheno adducts in humans may be controlled, in part, by the activity of two DNA N-glycosylases, the alkylpurine DNA, N-glycosylase and the mismatch-specific thymine DNA N-glycosylase (25) . The measured levels of etheno DNA adducts suggest that individuals a, b, c, and d might have been relatively deficient for the repair of {epsilon}C (Fig. 2B)Citation and patients a and b (and to a lesser extent c and d) for the repair of {epsilon}A (Fig. 1B)Citation .

There was no correlation between the levels of endogenous DNA damage and the age or sex of the patients or the postmortem interval (Table 1)Citation . All of the individuals examined in this study suffered from common illnesses associated with an increased oxidative stress/LPO, such as cancer (38) , atherosclerosis (39) , diabetes (40) , liver cirrhosis [see Fiorelli et al. (41) for alcoholic cirrhosis and Jain et al. (42) for hepatitis C virus-induced cirrhosis], liver steatosis, chronic obstructive pulmonary disease, and other lung diseases (43 , 44) , acute respiratory distress syndrome (45) , sepsis (46) , intestinal ischemia (47) , renal insufficiency (48) . Patient k had been exposed to asbestos, which is known to induce oxidative stress. However, in most cases, there was no correlation between the clinical/autopsy record of the patient and high levels of etheno adducts or ADLs in the affected tissue. Possible exceptions are discussed below.

With high levels of {epsilon}A and {epsilon}C in gray matter of the cerebrum, patient b had a metastatic melanoma and received palliative irradiation of the brain (five doses of 4 Gy each). He died 2 months later. It has been reported that {gamma}-irradiation of DNA in vitro leads to the formation of 3'-phosphoglycoaldehyde residues in DNA, which react in situ with nucleobases, yielding etheno adducts (49) . This could be an alternative pathway for the formation of ethenobases in vivo. Although etheno adducts have been shown to be cleaved from DNA in vitro through the base excision repair pathway (50) , several studies in vivo suggest that they are relatively persistent in DNA under certain circumstances (24 , 51 , 52) .

High levels of {epsilon}C were found in gray matter of the cerebrum from patient c, who suffered from a subdural hematoma, a condition that could possibly lead to an increased oxidative stress in the brain. Experimental traumatic injury to the brain of rodents leads to increased LPO (53) and increased levels of HNE (54) in the brain. Patient c also cumulated several diseases associated with increased oxidative stress/LPO: atherosclerosis, diabetes, pneumonia, and renal insufficiency. In a rat model, chronic renal failure resulted in an increased oxidative stress in the gray matter of the cerebrum (55) . There was no obvious factor in the clinical data that could explain the high amounts of etheno adducts detected in the brain white matter of patients a, d, and e. These patients suffered from liver cirrhosis (a, d) or from peripheral arterial occlusive disease (e), conditions that may be associated with systemic oxidative stress/LPO. In addition, patients a and d may have had a lower capacity for repairing {epsilon}C, as suggested by Fig. 2BCitation .

Patient d, who was diagnosed with a colon adenoma, had relatively high levels of {epsilon}A and {epsilon}C in DNA from colon tissue, but not from colon mucosa. Increased levels of {epsilon}A and {epsilon}C have been detected in colonic epithelia (polyps) of familial adenomatous polyposis patients and in colon tissue proximal to colon carcinoma (12) . It should be noted that, in the eight patients examined in the present study, we found, on the average, slightly higher (1.5-fold) levels of {epsilon}A and {epsilon}c in colon tissue (i.e., including the epithelial and muscular layers) as compared with colon mucosa. M1G, the major DNA adduct of malondialdehyde (another reactive aldehyde produced by LPO), is increased in the colon mucosa from patients with colon adenoma (56) . However, the level of M1G in colorectal mucosa is also affected by diet (56) . Dietary habits were not recorded for the present study, but there is the possibility that different dietary habits might explain why high amounts of {epsilon}A and {epsilon}C were found in colon DNA from patient d but not from patient e, who also suffered from colon adenoma.

Two patients, a and d, who suffered from liver cirrhosis, exhibited slightly elevated levels of {epsilon}C in their hepatic DNA. Liver cirrhosis (either of alcoholic or viral origin) is associated with stimulation of oxidative stress/LPO (41 , 42) , which could be a cause for these elevated levels of etheno DNA adducts. However, in a previous analysis of three liver samples from cirrhotic patients, no increase in the {epsilon}C/C ratio was found, compared with normal hepatic tissue; one cirrhotic liver sample contained a higher level of {epsilon}A than the two others and than the normal liver samples (6) . More recently, Nair et al. detected increased amounts of {epsilon}A in liver parenchyma from cirrhotic individuals.6 Administration of ethanol to rats induced the formation of both {epsilon}A and {epsilon}C in liver DNA (16) .

There is no known factor in the clinical data that could be associated with the high levels of etheno adducts measured in liver DNA of individual b and in lung DNA of individual a. As suggested above, lower DNA repair activities might be involved in part.

Patient h had very high levels of ADLs in colon DNA, but was not diagnosed with any disease of the colon. Other cases of slightly elevated levels of ADLs (Fig. 3A)Citation cannot be correlated with the clinical data, except the gray matter of the cerebrum of patient b and lung of individual l. Patient b, who also had high levels of {epsilon}C in the gray matter of the cerebrum (see above), suffered from a melanoma metastatic to the brain and had received radiotherapy before death. Patient l had acute alveolar injury of the lungs.

To conclude, these data demonstrate tissue-specific variations in the steady-state levels of endogenous DNA damage, both for etheno adducts and ADLs. In addition, interindividual differences in background amounts of etheno adducts may be determined, in part, by DNA repair activities. As expected, there was a strong correlation between levels of {epsilon}A and levels of {epsilon}C, two DNA lesions that arise through a common pathway, LPO (Fig. 3)Citation . The mean ratio of {epsilon}A/A to {epsilon}C/C was 0.49, which is similar to the ratio of 0.50 measured in DNA treated in vitro with 4-hydroxy-2,3-epoxynonanal (data not shown). No correlation was found in any of the tissues examined between amounts of ethenobases and amounts of ADLs.


   Acknowledgments
 
The authors thank the excellent technical assistance of G. Brun and A-M. Camus.


   Footnotes
 
Grant support: Grant ENV4-CT97-0505 from the European Union (to A. B.), the Foundation for Promotion of Cancer Research, Japan (to H. O.), and NIH Grants P42ES05948, R42ES11746, P30-CA16086, and P30-ES10126 (to J. A. S.).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Alain Barbin, The Unit of Endogenous Cancer Risk Factors, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon cedex 08, France. Phone: 33-4-72-73-85-38; Fax: 33-4-72-73-80-88; E-mail: barbin{at}iarc.fr

4 The abbreviations used are: LPO, lipid peroxidation; HNE, trans-4-hydroxy-2-nonenal; PUFA, polyunsaturated fatty acid; {epsilon}A, 1,N6-ethenoadenine; {epsilon}C, 3,N4-ethenocytosine; M1G, pyrimido[1,2-a]purin-10(3H)-one; AP, apurinic/apyrimidinic; ARP, aldehyde reactive probe; ADL, aldehydic DNA lesion. Back

5 Asakura, S. and Swenberg, J. A. manuscript in preparation. Back

6 Frank, A., Seitz, H. K., Frank, N., Bartsch, H., and Nair, J., manuscript in preparation. Back

Received 5/ 9/03; revised 7/25/03; accepted 7/30/03.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Migliore L., Coppede F. Genetic and environmental factors in cancer and neurodegenerative diseases. Mutat. Res., 512: 135-153, 2002.[Medline]
  2. Olinski R., Gackowski D., Foksinski M., Rozalski R., Roszkowski K., Jaruga P. Oxidative DNA damage: assessment of the role in carcinogenesis, atherosclerosis, and acquired immunodeficiency syndrome. Free Radic. Biol. Med., 33: 192-200, 2002.[Medline]
  3. Wallace S. S. Biological consequences of free radical-damaged DNA bases. Free Radic. Biol. Med., 33: 1-14, 2002.[Medline]
  4. Wilson D. M., III, Barsky D. The major human abasic endonuclease: formation, consequences and repair of abasic lesions in DNA. Mutat. Res., 485: 283-307, 2001.[Medline]
  5. Nath R. G., Chung F. L. Detection of exocyclic 1,N2-propanodeoxyguanosine adducts as common DNA lesions in rodents and humans. Proc. Natl. Acad. Sci. USA, 91: 7491-7495, 1994.[Abstract/Free Full Text]
  6. Nair J., Barbin A., Guichard Y., Bartsch H. 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytine in liver DNA from humans and untreated rodents detected by immunoaffinity/32P-postlabeling. Carcinogenesis (Lond.), 16: 613-617, 1995.[Abstract/Free Full Text]
  7. Chung F. L., Zhang L., Ocando J. E., Nath R. G. Role of 1, N2-propanodeoxyguanosine adducts as endogenous DNA lesions in rodents and humans Singer B. Bartsch H. eds. . Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis. IARC Scientific Publ. No. 150, 45-54, IARC Lyon, France 1999.
  8. Marnett L. J. Chemistry and biology of DNA damage by malondialdehyde Singer B. Bartsch H. eds. . Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis. IARC Scientific Publ. No. 150, 17-27, IARC Lyon, France 1999.
  9. Barbin A. Etheno-adduct-forming chemicals: from mutagenicity testing to tumor mutation spectra. Mutat. Res., 462: 55-69, 2000.[Medline]
  10. Yang I. Y., Chan G., Miller H., Huang Y., Torres M. C., Johnson F., Moriya M. Mutagenesis by acrolein-derived propanodeoxyguanosine adducts in human cells. Biochemistry, 41: 13826-13832, 2002.[Medline]
  11. Nair J., Carmichael P. L., Fernando R. C., Phillips D. H., Strain A. J., Bartsch H. Lipid peroxidation-induced etheno-DNA adducts in the liver of patients with the genetic metal storage disorders Wilson’s disease and primary hemochromatosis. Cancer Epidemiol. Biomarkers Prev., 7: 435-440, 1998.[Abstract]
  12. Schmid K., Nair J., Winde G., Velic I., Bartsch H. Increased levels of promutagenic etheno-DNA adducts in colonic polyps of FAP patients. Int. J. Cancer, 87: 1-4, 2000.[Medline]
  13. Hagenlocher T., Nair J., Becker N., Korfmann A., Bartsch H. Influence of dietary fatty acid, vegetable, and vitamin intake on etheno-DNA adducts in white blood cells of healthy female volunteers: a pilot study. Cancer Epidemiol. Biomarkers Prev., 10: 1187-1191, 2001.[Abstract/Free Full Text]
  14. Nair J., Gal A., Tamir S., Tannenbaum S. R., Wogan G. N., Bartsch H. Etheno adducts in spleen DNA of SJL mice stimulated to overproduce nitric oxide. Carcinogenesis (Lond.), 19: 2081-2084, 1998.[Abstract/Free Full Text]
  15. Nair J., Sone H., Nagao M., Barbin A., Bartsch H. Copper-dependent formation of miscoding etheno-DNA adducts in the liver of Long Evans cinnamon (LEC) rats developing hereditary hepatitis and hepatocellular carcinoma. Cancer Res., 56: 1267-1271, 1996.[Abstract/Free Full Text]
  16. Navasumrit P., Ward T. H., O’Connor P. J., Nair J., Frank N., Bartsch H. Ethanol enhances the formation of endogenously and exogenously derived adducts in rat hepatic DNA. Mutat. Res., 479: 81-94, 2001.[Medline]
  17. Bartsch H., Nair J. New DNA-based biomarkers for oxidative stress and cancer chemoprevention studies. Eur. J. Cancer, 36: 1229-1234, 2000.
  18. Lindahl T., Nyberg B. Rate of depurination of native deoxyribonucleic acid. Biochemistry, 11: 3610-3618, 1972.[Medline]
  19. Nakamura J., Walker V. E., Upton P. B., Chiang S. Y., Kow Y. W., Swenberg J. A. Highly sensitive apurinic/apyrimidinic site assay can detect spontaneous and chemically induced depurination under physiological conditions. Cancer Res., 58: 222-225, 1998.[Abstract/Free Full Text]
  20. Loeb L. A., Preston B. D. Mutagenesis by apurinic/apyrimidinic sites. Annu. Rev. Genet., 20: 201-230, 1986.[Medline]
  21. Breen A. P., Murphy J. A. Reactions of oxyl radicals with DNA. Free Radic. Bio. Med., 18: 1033-1077, 1995.
  22. Nakamura J., Swenberg J. A. Aldehyde reactive probe-slot blot assay to quantitate DNA lesions caused by oxidative stress Hensley K. Floyd R. A. eds. . Methods in Biological Oxidative Stress, 109-115, Humana Press Totowa, NJ 2003.
  23. Nakamura J., La D. K., Swenberg J. A. 5'-Nicked apurinic/apyrimidinic sites are resistant to ß-elimination by ß-polymerase and are persistent in human cultured cells after oxidative stress. J. Biol. Chem., 275: 5323-5328, 2000.[Abstract/Free Full Text]
  24. Ciroussel F., Barbin A., Eberle G., Bartsch H. Investigations on the relationship between DNA ethenobase adduct levels in several organs of vinyl chloride-exposed rats and cancer susceptibility. Biochem. Pharmacol., 39: 1109-1113, 1990.[Medline]
  25. Speina E., Zieliñska M., Barbin A., Gackowski D., Kowalewski J., Graziewicz M. A., Siedlecki J. A., Oliñski R., Tudek B. Decreased repair activities of 1,N6-ethenoadenine and 3,N4-ethenocytosine in lung adenocarcinoma patients. Cancer Res., 63: 4351-4357, 2003.[Abstract/Free Full Text]
  26. Kristofikova Z., Majer E., Fales E., Pekny I., Klaschka J. Effects of aging and dementia on the levels of thiobarbituric-acid-reactive products stimulated by L-glutamic acid in human autopsy and biopsy brain tissue. Dement. Geriatr. Cogn. Disord., 9: 6-12, 1998.
  27. Nakamura J., Swenberg J. A. Endogenous apurinic/apyrimidinic sites in genomic DNA of mammalian tissues. Cancer Res., 59: 2522-2526, 1999.[Abstract/Free Full Text]
  28. Barbin A. Role of etheno DNA adducts in carcinogenesis induced by vinyl chloride in rats Singer B. Bartsch H. eds. . Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis. IARC Scientific Publ. No. 150, 303-313, IARC Lyon, France 1999.
  29. Pratico D. Oxidative imbalance and lipid peroxidation in Alzheimer’s disease. Drug Dev. Res., 56: 446-451, 2002.
  30. Qi K., Hall M., Deckelbaum R. J. Long-chain polyunsaturated fatty acid accretion in brain. Curr. Opin. Clin. Nutr. Metab. Care, 5: 133-138, 2002.[Medline]
  31. El Ghissassi F., Barbin A., Nair J., Bartsch H. Formation of 1,N6-ethenoadenine and 3,N4-ethenocytosine by lipid peroxidation products and nucleic acid bases. Chem. Res. Toxicol., 8: 278-283, 1995.[Medline]
  32. Pan J., Chung F. L. Formation of cyclic deoxyguanosine adducts from {omega}-3 and {omega}-6 polyunsaturated fatty acids under oxidative conditions. Chem. Res. Toxicol., 15: 367-372, 2002.[Medline]
  33. Yavin E., Brand A., Green P. Docosahexaenoic acid abundance in the brain: a biodevice to combat oxidative stress. Nutr. Neurosci., 5: 149-157, 2002.[Medline]
  34. Kikugawa K. Effect of {omega}-6 and {omega}-3 fatty acid supplementation on rat tissue lipid peroxidation. World Rev. Nutr. Diet., 88: 28-34, 2001.[Medline]
  35. Noda Y., McGeer P. L., McGeer E. G. Lipid peroxide distribution in brain and the effect of hyperbaric oxygen. J. Neurochem., 40: 1329-1332, 1983.[Medline]
  36. Kristofikova Z., Klaschka J., Tejkalova H. Effect of aging on lipid peroxide levels induced by L-glutamic acid and estimated by means of a thiobarbituric acid test in rat brain tissue. Exp. Gerontol., 30: 645-657, 1995.[Medline]
  37. Liu J., Wang X., Shigenaga M. K., Yeo H. C., Mori A., Ames B. N. Immobilization stress causes oxidative damage to lipid, protein, and DNA in the brain of rats. FASEB J., 10: 1532-1538, 1996.[Abstract]
  38. Marnett L. J. Oxyradicals and DNA damage. Carcinogenesis (Lond.), 21: 361-370, 2000.[Abstract/Free Full Text]
  39. Aviram M. Review of human studies on oxidative damage and antioxidant protection related to cardiovascular diseases. Free Radic. Res., 33 (Suppl.): S85-S97, 2000.
  40. Bonnefont-Rousselot D., Bastard J. P., Jaudon M. C., Delattre J. Consequences of the diabetic status on the oxidant/antioxidant balance. Diabetes Metab., 26: 163-176, 2000.[Medline]
  41. Fiorelli G., De Feo T. M., Duca L., Tavazzi D., Nava I., Fargion S., Cappellini M. D. Red blood cell antioxidant and iron status in alcoholic and nonalcoholic cirrhosis. Eur. J. Clin. Investig., 32 (Suppl. 1): 21-27, 2002.
  42. Jain S. K., Pemberton P. W., Smith A., McMahon R. F., Burrows P. C., Aboutwerat A., Warnes T. W. Oxidative stress in chronic hepatitis C: not just a feature of late stage disease. J. Hepatol., 36: 805-811, 2002.[Medline]
  43. Morrow J. D., Roberts L. J. The isoprostanes: their role as an index of oxidant stress status in human pulmonary disease. Am. J. Respir. Crit. Care Med., 166: S25-S30, 2002.
  44. Bhalla D. K. Oxidative effects and inflammatory responses in the lung. J. Toxicol. Environ. Health Part A, 61: 3-8, 2000.[Medline]
  45. Gutteridge J. M., Mitchell J. Redox imbalance in the critically ill. Br. Med. Bull., 55: 49-75, 1999.[Abstract/Free Full Text]
  46. Taylor D. E., Piantadosi C. A. Oxidative metabolism in sepsis and sepsis syndrome. J. Crit. Care, 10: 122-135, 1995.[Medline]
  47. Thomson A., Hemphill D., Jeejeebhoy K. N. Oxidative stress and antioxidants in intestinal disease. Dig. Dis., 16: 152-158, 1998.[Medline]
  48. Annuk M., Zilmer M., Lind L., Linde T., Fellstrom B. Oxidative stress and endothelial function in chronic renal failure. J. Am. Soc. Nephrol., 12: 2747-2752, 2001.[Abstract/Free Full Text]
  49. Jones W. R., Dedon P. C. DNA oxidation as source of endogenous electrophiles: formation of ethenoadenine adducts in {gamma}-irradiated DNA. J. Am. Chem. Soc., 121: 9231-9232, 1999.
  50. Saparbaev M., Laval J. Enzymology of the repair of etheno adducts in mammalian cells and in Escherichia coli. Singer B. Bartsch H. eds. . Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis. IARC Scientific Publ. No. 150, 249-261, IARC Lyon, France 1999.
  51. Guichard Y., El Ghissassi F., Nair J., Bartsch H., Barbin A. Formation and accumulation of DNA ethenobases in adult Sprague-Dawley rats exposed to vinyl chloride. Carcinogenesis (Lond.), 17: 1553-1559, 1996.[Abstract/Free Full Text]
  52. Morinello E. J., Ham A. J., Ranasinghe A., Nakamura J., Upton P. B., Swenberg J. A. Molecular dosimetry and repair of N2,3-ethenoguanine in rats exposed to vinyl chloride. Cancer Res., 62: 5189-5195, 2002.[Abstract/Free Full Text]
  53. Pratico D., Reiss P., Tang L. X., Sung S., Rokach J., McIntosh T. K. Local and systemic increase in lipid peroxidation after moderate experimental traumatic brain injury. J. Neurochem., 80: 894-898, 2002.[Medline]
  54. Zhang D., Dhillon H. S., Mattson M. P., Yurek D. M., Prasad R. M. Immunohistochemical detection of the lipid peroxidation product 4-hydroxynonenal after experimental brain injury in the rat. Neurosci. Lett., 272: 57-61, 1999.[Medline]
  55. Deng G., Vaziri N. D., Jabbari B., Ni Z., Yan X. X. Increased tyrosine nitration of the brain in chronic renal insufficiency: reversal by antioxidant therapy and angiotensin-converting enzyme inhibition. J. Am. Soc. Nephrol., 12: 1892-1899, 2001.[Abstract/Free Full Text]
  56. Leuratti C., Watson M. A., Deag E. J., Welch A., Singh R., Gottschalg E., Marnett L. J., Atkin W., Day N. E., Shuker D. E., Bingham S. A. Detection of malondialdehyde DNA adducts in human colorectal mucosa: relationship with diet and the presence of adenomas. Cancer Epidemiol. Biomarkers Prev., 11: 267-273, 2002.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Barbin, A.
Right arrow Articles by Swenberg, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barbin, A.
Right arrow Articles by Swenberg, J. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation