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Laboratory of Chemical Carcinogenesis, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation [P. G., S. K., S. A. K.], and Department of Hygiene and Epidemiology, University of Athens Medical School [E. S., K. K.], Athens 11635, Greece
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Biological evidence of human exposure to methylating agents comes from studies that have demonstrated the presence of methylated DNA adducts in human tissues. For example, in a number of pilot-scale studies, N7-methylguanine, the major DNA adduct formed by methylating agents, has been detected by 32P-postlabeling in the DNA of tissues (mostly lung) of individuals suffering general environmental exposure (Refs. 3, 4, 5, 6, 7, 8, 9, 10 ; reviewed in Ref. 11 ). The adduct was usually found in the range of 0.1–1 µmol N7-meG/mol G, levels that are high by comparison to most other DNA adducts found in human DNA. Furthermore, the frequency of adduct-positive samples was also high (usually ranging from 30 to 100%), suggesting that exposure to methylating agents is common. However, no systematic studies of the epidemiology of N7-meG have been reported, and evidence that its occurrence in human DNA is related to tobacco smoke exposure is limited and contradictory (4 , 7 , 10) .
A few studies have also examined the presence in human tissues of additional, biologically important methylated DNA lesions, such as the directly miscoding adducts O6-meG and O4 -meT. In pilot-scale studies, O4 -meT was detected in human liver DNA (but not in WBC DNA) at levels <1 µmol/mol T (12) . Using an HPLC/radioimmunoassay method with limit of detection of 0.5 µmol O6-meG/mol G, Foiles et al. (13) found this adduct in 5 samples of human placenta of 20 examined, in the range 0.6 to 1.6 µmol/mol G, whereas in more recent investigations of similar size, utilizing more sensitive assays, the same adduct has been found in a large proportion of human WBCs (range, 7–46 nmol/mol G) and liver (range, 110–670 nmol/mol G) DNA samples (14 , 15) . As in the case of N7-meG, these studies suggested that exposure to methylating agents may be commonplace, but their limited scale did not permit the investigation of the origin of the adducts. A role for endogenously formed N-nitroso compounds is suggested by the observation of increased incidence of O6-meG in DNA from the urinary bladder of individuals infected with schistosomiasis (which leads to inflammation and concomitant production of nitric oxide and related nitrosating species; Ref. 16 ) and by the discovery of O6-meG in esophageal DNA of individuals in China with high dietary intake of nitrates (17) . In a large-scale investigation of the epidemiology of O6-meG, involving examination of blood leukocyte DNA of 407 individuals drawn from 17 regions worldwide, O6-meG was found in 21 samples at levels ranging from the limit of detection of the assay used (0.08 µmol/mol G) up to 0.3 µmol/mol G (18) . In this study, adducts were found more frequently among samples derived from regions with higher consumption of nitrate-treated foods and incidence of gastric cancer and from individuals with low levels of serum pepsinogen A (a marker of chronic atrophic gastritis). These observations were consistent with the hypothesis that agents formed through intragastric nitrosation contributed significantly to methylated adduct formation and implied that such adducts might have biological significance.
The suggestion that endogenous nitrosation (a process that occurs to a greater or lesser extent in everyone) may be an important source of DNA methylation implies that low levels of methylated adducts in human DNA may be commonplace, even in individuals with conditions that are not associated with high endogenous nitrosation (i.e., chronic inflammation or high nitrate intake). To further test this suggestion, an assay of improved sensitivity was developed and used for the investigation of the presence of O6-meG in maternal and cord blood leukocyte DNA obtained from a group of healthy women living in Greece, a region known not to be associated with high consumption of nitrate-containing foods.
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Materials and Methods |
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Samples of venous and cord blood (10 ml) were collected at the time of birth and stored at room temperature until processing, which took place within 6 h of collection. No anaesthetic treatment had been given to any up to the time of blood collection. On the day after delivery, they underwent an interview, in the context of which a questionnaire was completed giving information on, among other matters: (a) exposure to products of fuel combustion (place of residence in relation to traffic density, type of home heating and cooking facilities); (b) dietary habits, with particular attention to consumption of fruits and vegetables and smoked or other nitrate-treated meat or fish; (c) active or passive smoking (habitual smoking status, number of cigarettes smoked during the last 24 h, residence with a smoker, number of cigarettes smoked in her presence during the last 24 h); (d) consumption of vitamin supplements; and (e) family history of cancer. Three of the women left the maternity clinic before this interview could be undertaken.
Total blood DNA was extracted by standard methods involving multiple proteinase K and RNase 1 treatments and phenol/chloroform extractions (19) . Lymphocytes for AGT analysis were isolated using Ficoll gradient (Lymphoprep; NYCOMED Pharma, Oslo, Norway), according to the manufacturer’s procedure.
Assay for O6-meG.
DNA adduct analysis was carried out using precautions to minimize the
risk of cross-contamination, such as avoiding the handling of known
methylated DNA samples in the same area of the laboratory and using
wherever possible single-use, disposable plasticware. A modification of
the competitive repair assay for O6-meG
(20)
was used which had a higher sensitivity than the
classical assay used in our previous studies. This assay involves the
use of an excess of the repair enzyme
O6-alkylguanine-DNA alkyltransferase from
Escherichia coli (the ada protein) to repair
O6-meG present in the DNA under examination,
followed by back-titration of the remaining enzyme using a synthetic
oligonucleotide containing a single residue of
O6-meG and 5'-end labeled with
[35S]-thiophosphate. The amount of unrepaired labeled
oligonucleotide is measured by immunoprecipitation with rabbit
anti-O6-meG antiserum, and the amount of
O6-meG is finally calculated from a standard
curve. This assay, which has a limit of detection of 0.5 fmol
O6-meG in up to 10 µg of DNA (thus permitting
the detection 80 nmol O6-meG/mol G) has been
extensively validated in studies of animals treated with methylating
agents as well as in studies of humans treated with methylating
cytostatic drugs (19
, 21
, 22) .
A modification introduced in the present case involved primarily the
use of labeled oligonucleotide of higher specific activity (achieved
through the HPLC separation of the 35S-thiophosphorylated
oligonucleotide from the unlabeled starting material), as well as a few
additional changes. After the 5'-end labeling of the oligonucleotide
with 
-35S-labeled ATP and T4 polynucleotide kinase, the
radioactive oligonucleotide was recovered free of unlabeled starting
material using anion exchange HPLC (monoQ HR5/5 column, gradient 0.2 to
0.8 M NaCl, pH 12, and flow 1 ml/min). The typical specific
activity of the resulting material was 2200 cpm/fmol (equal to the
specific activity of the -35S-labeled ATP), 7–15-fold
higher than that of the material used in the classical assay. Use of
this high specific activity oligomer in the competitive repair assay as
described previously (20)
, with AGT-pretreated (hence
O6-meG-free) DNA samples, was found to result
occasionally in a weak positive signal (equivalent to up to 0.4 fmol of
O6-meG), presumably caused by nonspecific
inhibitors of AGT present in the DNA. For this reason, an additional
modification was introduced to control for such nonspecific AGT
inhibition, based on the use the method of standard additions instead
of the commonly used standard curve method for adduct quantitation.
Thus, the assay was conducted as follows. Triplicate "test"
mixtures consisting of 1 µg of standard, methylated calf thymus DNA,
containing 0, 0.2, 0.4, or 0.6 fmol O6-meG, each
mixed with 10 µg of unknown DNA sample, plus a separate triplicate
"control" mixture consisting of 1 µg of unmethylated calf thymus
DNA plus 10 µg of the same unknown DNA, previously treated overnight
with a large excess AGT to remove all O6-meG and
repurified by phenol extraction, were prepared. To each mixture, AGT
(10 fmol) was added, and O6-meG repair was
allowed to proceed for 2 h at room temperature, followed by the
addition of the 35S-labeled oligonucleotide (7 fmol). After
further incubation overnight, any oligonucleotide remaining unrepaired
was finally immunoprecipitated, and its radioactivity was counted, as
described previously (20)
. The inhibition of
oligonucleotide repair caused by the O6-meG
present in the unknown DNA was evaluated by subtracting the amount of
radioactivity immunoprecipitated from the "control" mixture from
that immunoprecipitated from the test mixture containing the unknown
DNA without O6-meG-containing standard DNA. The
amount of adduct present in the unknown DNA was calculated by dividing
this difference by the slope of the "test" standard addition curve.
Typical standard addition curves for a pair of maternal and cord blood
DNA are shown in Fig. 1
. The intra- and interassay variabilities were less than 7 and 10%,
respectively. The limit of sensitivity of the assay reached 0.10 fmol
O6-meG in 10 µg of DNA (16 nmol
O6-meG/mol G).
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Statistical Analysis.
O6-Methylguanine adduct levels and AGT levels
were approximately normally distributed, as indicated by application of
the Kolmogorov-Smirnov normality test, and consequently were analyzed
using Student’s t test (paired and two-sample), ANOVA, and
multiple linear regression models.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The mean value for the adduct levels in maternal blood DNA was
statistically significantly higher than that in cord blood DNA
(P = 0.047; Student’s paired t test). A
strong correlation between adduct levels in maternal and cord blood DNA
was observed (r2 = 0.47; P < 0.001; Fig. 3
).
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. The mean values were 8.32 fmol/µg DNA (SD, 2.91 fmol/µg DNA) and
7.43 fmol/µg DNA (SD, 2.2 fmol/µg DNA) for maternal and cord blood
lymphocytes, respectively. Maternal lymphocytes appeared to have higher
AGT activity than cord blood lymphocytes, although the difference was
not statistically significant (P = 0.089; Student’s
paired t test). No correlation was found between AGT
activities in maternal and the corresponding cord blood lymphocytes
(r2 = 0.005; P = 0.634),
nor was there a correlation between AGT and adduct levels in either
maternal or cord WBC DNA (r2 = 0.026,
P = 0.37; and r2 < 0.001,
P = 0.93, respectively).
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). This result was not altered when other parameters that might affect the
level of residence air pollution (distance from streets with heavy
traffic and type of indoor heating and cooking) were taken into
consideration (results not shown). Similarly, no effect was observed
for any of the other parameters examined, including habitual active or
passive smoking status or corresponding recent exposures; consumption
of fruits and vegetables, fruit juices, or smoked foods; or family
history of cancer (Table 1)
. A nonstatistically significant difference
toward lower adduct levels, especially in cord blood DNA, was observed
in women taking vitamin supplements as compared with those not taking
such supplements (P = 0.11, t test). This
difference remained evident when a multiple linear regression model was
employed utilizing adduct levels as the dependent variable and
residence air pollution, exposure to active or passive smoking,
consumption of fruits and vegetables, fruit juices, and vitamin
supplements as independent variables (Table 2)
. After adjustment for the remaining model variables, those consuming
vitamin supplements had on average 27 nmol
O6-meG/mol G in maternal WBC DNA
(P = 0.08) and 26 nmol
O6-meG/mol G in cord WBC DNA less than those not
taking such supplements (P = 0.15).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-fold lower than the limit of sensitivity of the version
reported previously of the assay. Although the modified assay is
significantly more complex and requires substantially larger amounts of
DNA, its availability constitutes a significant development that may
facilitate future studies of the epidemiology of
O6-meG. In a previous study, we found a higher frequency of O6-meG in the DNA of individuals drawn from regions known to be associated with high consumption of nitrate-treated foods as well as with high incidence of gastric cancer (18) . In that study, none of 74 WBC DNA samples derived from Greek individuals was found to contain O6-meG above the limit of detection of the assay used on that occasion. Greece is a region in which the prevalent diet does not include frequent consumption of nitrate-treated foods or other items likely to be rich in N-nitroso compounds, and additionally it has a relatively low incidence of most cancers, including gastric cancer (23 , 24) .
Use of a more sensitive assay in the presently reported study resulted in the same adduct being detected in most of 36 pairs of maternal and cord WBC DNA samples examined, almost always at levels below the limit of detection of the previously used version of the assay. The frequent observation of O6-meG is compatible with the hypothesis described in the "Introduction" of this report, i.e., that endogenous nitrosation, a phenomenon known to occur even in individuals without excessive exposure to nitrate or other nitrosation precursors, gives rise to a ubiquitous background level of DNA methylation damage. Such a background may be increased by exposure to unusually large amounts of nitrosation precursors or to preformed N-nitroso compounds themselves, situations that have been epidemiologically associated with increased incidence of various types of cancer (25) .
The levels of O6-meG in peripheral WBC DNA
observed in the present study are comparable with those reported by
others (see "Introduction"). Adduct levels in cord blood DNA were
measured for the first time in the present investigation. They were
found to be lower than, and showed a strong correlation with, those in
maternal blood DNA, suggesting transplacental exposure of the fetus to
methylating agents circulating in the mother’s blood. Methylating
N-nitroso compounds such as NDMA, MNU, and the
tobacco-specific nitrosamine NNK are active transplacental carcinogens
in rodents (26, 27, 28, 29)
, and they can cause DNA methylation in
rodent embryonic tissues (30, 31, 32, 33)
. However, information on
the relative levels of DNA damage induced specifically in maternal and
cord blood DNA is available only for NDMA. In a study in patas monkeys,
Chhabra et al. (34)
have shown that a single
dose of NDMA gave rise to O6-meG in cord blood
leukocytes at levels 25% of those seen in maternal WBC. Thus, the
relative adduct levels observed in the present study are compatible
with their being derived from NDMA.
No correlation was observed between O6-meG and AGT levels. This is in line with similar observations in patients accumulating much large levels of O6-meG as a result of chemotherapy with methylating drugs such as procarbazine or dacarbazine, where only in individuals with very low levels of AGT was a trend toward increased adduct accumulation observed (Refs. 19 , 22 , and 35 ; reviewed in Ref. 36 ). These observations suggest that AGT variation within the range usually observed in human populations may not substantially affect O6-meG accumulation.
Studies in humans treated with methylating chemotherapeutic drugs have shown that O6-meG in human WBCs is relatively short-lived; its half-life is approximately 22–25 h (19 , 35) . This implies that the adduct levels measured in the present study were probably determined primarily by recent exposure (previous 24 h). For this reason, an effort was to include among the parameters utilized in the statistical analysis factors likely to reflect recent exposure to methylating agents or their precursors. Given that the study subjects were pregnant women who had spent the last 24 h prior to sample donation in the same maternity hospital, suffering similar ambient and dietary exposures during this time, the most important additional factors to control for had to do with recent smoking exposure. This was done by including in our analysis information on habitual active or passive smoking, as well as on cigarettes smoked by or in the presence of each subject during the previous 24 h. However, neither these nor any of the other questionnaire parameters examined showed a statistically significant correlation with adduct levels.
The only parameter that showed a consistent and nearly statistically
significant trend toward being associated with lower adduct levels, in
both maternal and cord WBC DNA, was intake of vitamin supplements
(Tables 1
and 2)
. In all cases the supplements consisted of vitamin C
alone or in combination with other vitamins. Vitamin C is known to be a
potent inhibitor of nitrosation reactions in vitro and
in vivo (37)
, especially at high doses such as
those associated with the intake of vitamin supplements
(38)
. Thus, the observed association is compatible with,
and provides plausibility to, the suggestion that methylation damage
may arise via endogenous generation of methylating N-nitroso
compounds. It is notable that maternal consumption of vitamin
supplements has been reported recently in two studies to be associated
with a protective effect against childhood brain tumours (39
, 40)
. In the latter study, maternal consumption of vitamin
supplements appeared to reduce childhood brain cancer risks associated
with consumption of cured meats during pregnancy, an observation
compatible with their acting to block endogenous nitrosation reactions.
A similar protective effect of vitamin supplements against cured
meat-associated brain cancer risks has also been reported in a study of
adult brain cancer in women (41)
. Finally, vitamin
supplement consumption has also been reported to reduce the risk of
brain cancer in men (42)
. These epidemiological
observations, in combination with our observation of a possible
protective effect of vitamin supplements against the induction of the
precarcinogenic DNA adduct O6-meG in both
maternal and cord blood DNA, provides some support for the hypothesis
that endogenous nitrosation processes may play a role in the etiology
of adult or childhood brain carcinogenesis.
The identity of the methylating agent(s) responsible for the induction of the observed adducts is unknown. The well-known ability of SN 1-type methylating agents to induce relatively high levels of O-methylation makes it likely that the agent in question belongs to this chemical category. Animal studies have shown that NDMA is, on a per orally administered dose basis, by far the most efficient agent capable of generating O6-meG in blood cell DNA when compared with other known methylating agents (e.g., MNU and NNK; Refs. 21 and 43 ).4 Hence, given the known endogenous formation of NDMA in the human body, as indicated by its presence in gastric juice and its excretion in urine (44 , 45) , this nitrosamine seems a plausible candidate for giving rise to the observed adducts. This possibility is also compatible with the available information, already mentioned, on the relative ability of NDMA to methylate maternal and cord blood leukocyte DNA of patas monkeys (34) .
In conclusion, our demonstration of the presence of the precarcinogenic adduct O6-meG in maternal and cord leukocyte DNA of a large proportion of women not thought to have suffered high exposure to known methylating agents or N-nitroso compounds or their precursors indicates that DNA methylation constitutes a significant genotoxic burden for the general population. Furthermore, the observation of a possible protective effect of intake of vitamin C supplements provides limited support for the hypothesis that endogenous nitrosation processes may contribute significantly to this burden.
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Acknowledgments |
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Footnotes |
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1 This work was partially financed by the European
Union under Contract EV5V-CT92-0118. 
2 To whom requests for reprints should be
addressed, at Laboratory of Chemical Carcinogenesis, Institute of
Biological Research and Biotechnology, National Hellenic Research
Foundation, 48 Vas. Constantinou Avenue, Athens 11635, Greece. Phone:
30-1-7273740; Fax: 30-1-7251827; E-mail: skyrt{at}eie.gr
3 The abbreviations used are:
O6-meG, O6-methylguanine;
O4-meT, O4-methylthymine;
NDMA, N-nitrosodimethylamine; MNU,
N-methyl-N-nitrosourea; NNK,
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; HPLC, high-performance
liquid chromatography; AGT, alkylguanine-DNA alkyltransferase.
4 Souliotis et al., unpublished
observations.
Received 7/28/99; revised 11/ 2/99; accepted 12/ 8/99.
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