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

Chunyan Zhao,1 and Kari Hemminki

Department of Biosciences at Novum, Karolinska Institute, 14157 Huddinge, Sweden

Abstract

Endogenous DNA damage is assumed to be a major contributor to aging and cancer. This study compares the steady-state levels of 7-methyl- and 7-(2-hydroxyethyl)-guanine DNA adducts in lymphocytes isolated from the younger (mean age 39.8 years) and the older (mean age 82.8 years) healthy subjects. Using a 32P-post-labelling method, these adducts were measured in lymphocyte DNA from a total of 34 subjects. The results show that the amount of both 7-methyl- and 7-(2-hydroxyethyl)-guanine adducts in the younger age group was similar to that in the older age group. Our findings show that at steady-state the levels of DNA alkylation products are independent of age, suggesting that endogenous DNA damage, through methylation or lipid peroxidation, and the repair of such damage may not be deficient in lymphocytes of older individuals.

Abbreviations: DMS, dimethylsulphate; ETO, ethylene oxide; HPLC, high-pressure liquid chromatography; TLC, thin-layer chromatography

Introduction

Cancer is a disease of old age, and the risk of carcinoma increases exponentially with age (1). According to the somatic mutation theory, the age dependence has been explained by accumulation of mutations in the tumour-initiating cell that then expand to a clinical cancer (13). Although the known mutation rates are sufficiently high to accommodate multiple hits in one cell, some researchers have proposed that aging conveys mechanisms that accelerate the accumulation of DNA damage and mutations. These include an age-dependent increase in oxidative damage to DNA and a decrease in DNA damage repair (46). However, human in vivo data supporting these notions are limited, even though humans are continuously exposed to DNA damaging agents from both endogenous and exogenous sources and it is assumed that endogenous DNA damage may be a major contributor to aging and cancer (7). It has been assumed that age-related factors such as differing rates of cell proliferation, abilities to activate and detoxify carcinogens and repair DNA damage may modulate individual susceptibility to chemical carcinogenesis (5,8). As an indicator of DNA damage, carcinogen–DNA adducts are an informative biomarker of age-related susceptibility, but their application has been limited to oxidative damage, I-compounds and 7-methyl-guanine in rodent models (7,913). The results await quantitative confirmation on humans and with chemically defined DNA adducts.

Alkylating agents react predominantly with the N-7 position of guanine (14). 7-Methyl-guanine adducts are induced exogenously by methylating agents such as N-nitroso compounds present in the environment and diet, and endogenously by the reactive S-adenosylmethionine present in the nucleus (15). Ethene is ubiquitous in the environment, arising from cigarette smoking and vehicle exhaust, and human exposure may give rise to 7-(2-hydroxyethyl)-guanine DNA adducts (16). Furthermore, ethene is formed endogenously from several possible sources including lipid peroxidation of unsaturated fats, oxidation of free methionine, oxidation of haemin in haemoglobin and metabolism by intestinal bacteria (17,18). These and other 7-alkyl-guanine adducts have labile glycosylic bonds that are subject to spontaneous depurination (14). In vivo, these adducts are removed from DNA via the base excision repair pathway, in which the modified bases are recognized by DNA glycosylases, leaving an abasic site (19). The resulting abasic sites are potentially mutagenic if left unrepaired (20). Recently, an age-dependent decline in the activity of the glycosylase was reported in human leukocytes in vitro (6). The purpose of the present study was to investigate the effect of age on 7-methyl- and 7-(2-hydroxyethyl)-guanine DNA adduct levels in healthy humans. Using the 32P-post-labelling technique, steady-state levels of these adducts were determined in lymphocyte DNA from the younger (mean age 39.8 years) and the older (mean age 82.8 years) individuals.

Materials and methods

Human subjects, lymphocyte preparation and DNA isolation
The study population included 34 healthy subjects, who were all non-smokers. Of the participants, 14 were younger than 50 years (mean age ± SD, 39.8 ± 7.2) and 20 were older than 80 years (mean age ± SD, 82.8 ± 3.3) (Table IGo).


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Table I. Mean levels of 7-methyl- and 7-(2-hydroxyethyl)-guanine (adducts per 107 nucleotides; mean ± SD) in lymphocytes of young and old healthy subjects
 
Blood samples of 20 ml were collected in heparinized sterile tubes. Lymphocytes were obtained by centrifugal separation in a Ficoll (Pharmacia Chemical Company, Sweden) gradient (21). DNA was isolated from the nuclei of lymphocytes by means of enzyme incubation and solvent extraction (22).

DNA adduct standards
7-Methyl- and 7-(2-hydroxyethyl)-deoxyguanosine-5-monophosphate adducts were prepared by reaction of the unmodified nucleotide with dimethylsulphate (DMS) and ethylene oxide (ETO), respectively, followed by purification by high-pressure liquid chromatography (HPLC). DNA standards for these two adducts were obtained by treating salmon testis DNA with DMS and ETO, respectively, as described elsewhere (23).

32P-Post-labelling and HPLC separation
The adduct levels were quantified using the 32P-post-labelling method, which was described elsewhere (23). Briefly, for each post-labelling assay, 10 µg DNA was digested and labelled. The post-labelling reaction was carried out in a total volume of 2.0 µl containing 2 pmol [{gamma}-32P]ATP, 40 mM bicine buffer, pH 9.6, 20 mM spermidine and 6 U T4 polynucleotide kinase. After labelling, the whole solution was applied on a thin-layer chromatography (TLC) plate and adducts were separated by chromatography in two dimensions. Subsequently, the adduct spot was extracted and analysed with a Beckman HPLC (Fullerton, CA) coupled with a UV and a Beckman 171 Radioisotope detector (Phenomenex, Torrance, CA). A 5 µ Prodigy 2.0 x 250 mm C18 reversed phase column was used for adduct separation.

Analysis of results
The in vitro modified DNA with DMS and ETO were labelled in parallel to each set of human DNA samples. Based on known adduct levels in these DNA standards, the recovery from DNA hydrolysis through all steps of the post-labelling procedure was found to be 25.0 ± 6% (n = 5) for 7-methyl-guanine and 12.0 ± 4% (n = 5) for 7-(2-hydroxyethyl)-guanine and was used to correct the levels of adducts in human DNA samples.

Results

Our previously described 32P-post-labelling/HPLC method to detect 7-methyl- and 7-(2-hydroxyethyl)-guanine DNA adducts was used in this study. Figure 1AGo shows a representative chromatogram of HPLC separation of a mixed standard of DMS-modified DNA and ETO-modified DNA. The retention times were ~17.0 min for 7-(2-hydroxyethyl)-guanine and 21.0 min for 7-methyl-guanine (as indicated by the arrows). The DNA standards were used as external standards for correction of determined adduct levels in human lymphocyte samples. The typical profile of HPLC separation of 7-methyl- and 7-(2-hydroxyethyl)-guanine adducts from lymphocyte DNA of one subject is shown in Figure 1BGo. The identities of these two adducts were established by their co-migration with the synthesized 7-methyl- and 7-(2-hydroxyethyl)-deoxyguanosine-5'-monophosphate standards used as UV markers (Figure 1BGo).



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Fig. 1. HPLC analysis of 7-(2-hydroxyethyl)-guanine (peak `a') and 7-methyl-guanine adducts (peak `b') in (A) in vitro DNA reacted with DMS and ETO; (B) lymphocyte DNA of one subject. `— — —' radioactivity, `...' UV. Note that the UV detector is installed 0.6 min after the radioactivity detector.

 
Table IGo lists the ages and levels of 7-methyl- and 7-(2-hydroxyethyl)-guanine adducts of the subjects. The presence of these two adducts was detected in all subjects. A similar amount of 7-methyl- and 7-(2-hydroxyethyl)-guanine adducts was found in the younger age group and the older age group. Thus, the mean levels of 7-methyl- and 7-(2-hydroxyethyl)-guanine in the younger age group were 1.0 and 3.8 adducts/107 nucleotides and in the older age they were 0.8 and 3.0 adducts/107 nucleotides, respectively. The level of 7-(2-hydroxyethyl)-guanine was ~4.0-fold higher than that of 7-methyl-guanine in both age groups. When 7-methyl- and 7-(2-hydroxyethyl)-guanine levels obtained from the same subjects were compared in the whole study group, no correlation was observed (r = 0.14). For both 7-methyl- and 7-(2-hydroxyethyl)-guanine adducts, the variation between the highest and the lowest adduct level between individuals was ~15-fold.

Discussion

Some DNA adducts have been studied in experimental animals in relation to aging, including 8-hydroxy-guanine (7), I-compounds (9), 7-methyl-guanine (24) and malondialdehyde–DNA adducts (10), and benzo[a]pyrene–DNA adducts have been studied in humans (25). In the case of 7-methyl-guanine, an increase of background levels of 7-methyl-guanine in senescent mice was reported (13). In the present study, we compare levels of 7-methyl- and 7-(2-hydroxyethyl)-guanine DNA adducts between two age groups of healthy subjects. The adduct levels were determined by TLC combined with a HPLC assay, which has been used previously to detect these adducts in smokers and non-smokers (26). When this method was compared with GC/MS in quantifying 7-(2-hydroxyethyl)-guanine from in vivo samples, a good qualitative agreement between methods with a correlation coefficient of 0.97 was obtained (27).

Our results show that the steady-state levels of 7-methyl- and 7-(2-hydroxyethyl)-guanine adducts in lymphocyte DNA were ~1.0 and ~ 3.5 adducts/107 nucleotides, respectively. These adducts have been detected in human tissues using various methods, including immunological assays, fluorescence techniques, mass spectrometry, 32P-post-labelling and electrochemical detection (26). So far the data obtained by various methods suggest that the background levels of 7-methyl-guanine adducts in human DNA range from 1 adduct/107 to >2 adducts/106 nucleotides, while 7-(2-hydroxyethyl)-guanine adduct levels appear to be <1 adduct/106 nucleotides (26). This is in conformity with our findings. Our results showed a 15-fold inter-individual variation for both 7-methyl- and 7-(2-hydroxyethyl)-guanine adduct levels between people. Previously, large inter-individual variation in 7-alkyl-guanine levels was also observed in human lung tissue (28). The large inter-individual difference in DNA adduct levels in humans can be due to the variation in the individual capacity to activate or inactivate carcinogens and repair DNA damage.

In the present study, no age-dependent change in the steady-state levels of 7-methyl- and 7-(2-hydroxyethyl)-guanine in human lymphocyte DNA was evident. The steady-state levels of DNA adducts reflect tissue-specific rates of adduct formation and removal (29). Therefore, the change of either the rate of formation or the rate of removal will influence the steady-state amounts of adducts. There are a number of endogenous sources for background exposure to alkylating agents. Thus, 7-methyl-guanine adducts are formed by endogenous methylating agents such as N-nitroso compounds and S-adenosylmethionine, whereas 7-(2-hydroxyethyl)-guanine adducts are endogenously formed by lipid peroxidation. The removal of these adducts occurs spontaneously because of adduct instability by dilution during cell turnover, and enzymatically by DNA repair processes. In two animal studies, levels of endogenous and induced 7-methyl-guanine levels were found to be higher in old compared with young mice (13,24). More recently, it was reported that lipid peroxidation in rats increased with age (30). The conflicting results concerning the change of 7-methyl-guanine levels during aging may be related to the different behaviours among tissues investigated. Thus, in animal studies, post-mitotic tissues, including liver, were used, whereas the present study employed human lymphocytes with continuous cell turnover. However, these methylation findings in animal experiments remained unexplained because neither S-adenosylmethionine levels nor 7-methyl-guanine repair activities were found to vary with age (13,24,31). The suggestion by Gaubatz and Tan (24) that 7-methyl-guanine accumulates in DNA may also be unlikely because of the short half-life in vivo, e.g. 3–5 days for 7-(2-hydroxyethyl)-guanine (32) and 2.5 days for 7-methyl-guanine (33).

In summary, our results show that the steady-state levels of 7-methyl- and 7-(2-hydroxyethyl)-guanine DNA adducts in lymphocytes of older healthy subjects were similar to those of the younger subjects. These results suggest that endogenous DNA damage, through methylation or lipid peroxidation, and the repair of such damage may not be deficient in lymphocytes of older individuals.

Notes

1 To whom correspondence should be addressed Email: chunyan.zhao{at}cnt.ki.se Back

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Received November 29, 2001; revised November 29, 2001; accepted November 30, 2001.