Carcinogenesis vol.18 no.11 pp.2071–2076, 1997
Presence and consequence of uracil in preneoplastic DNA from
folate/methyl-deficient rats
Igor P.Pogribny2, Levan Muskhelishvili1,
Barbara J.Miller and S.Jill James3
Division of Biochemical Toxicology, FDA–National Center for
Toxicological Research, Jefferson, AR 72079, 1Pathology Associates
International, National Center for Toxicological Research, Jefferson, AR
72079 and 2Department of Biochemistry and Molecular Biology, University
of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
3To
whom correspondence should be addressed
Uracil can arise in DNA by misincorporation of dUTP into
nascent DNA and/or by cytosine deamination in established
DNA. Based on recent findings, both pathways appear to
be promoted in the methyl-deficient model of hepatocarcinogenesis. A chronic increase in the ratio dUTP:dTTP with
folate/methyl deficiency can result in a futile cycle of
excision and reiterative uracil misincorporation leading to
premutagenic apyrimidinic (AP) sites, DNA strand breaks,
DNA fragmentation and apoptotic cell death. The progressive accumulation of unmethylated cytosines with chronic
methyl deficiency will increase the potential for cytosine
deamination to uracil and further stress uracil mismatch
repair mechanisms. Uracil is removed by a highly specific
uracil-DNA glycosylase (UDG) leaving an AP site that is
subsequently repaired by sequential action of AP endonuclease, 59-phosphodiesterase, a DNA polymerase and DNA
ligase. Since the DNA polymerases cannot distinguish
between dUTP and dTTP, an increase in dUTP:dTTP ratio
will promote uracil misincorporation during both DNA
replication and repair synthesis. The misincorporation
of uracil for thymine (5-methyluracil) may constitute a
genetically significant form of DNA hypomethylation distinct from cytosine hypomethylation. In the present study
a significant increase in the level of uracil in liver DNA as
early as 3 weeks after initiation of folate/methyl deficiency
was accompanied by parallel increases in DNA strand
breaks, AP sites and increased levels of AP endonuclease
mRNA. In addition, uracil was also detected within the
p53 gene sequence using UDG PCR techniques. Increased
levels of uracil in DNA implies that the capacity for uracil
base excision repair is exceeded with chronic folate/methyl
deficiency. It is possible that enzyme-induced extrahelical
bases, AP sites and DNA strand breaks interact to negatively affect the stability of the DNA helix and stress the
structural limits of permissible uracil base excision repair
activity. Thus substitution of uracil for thymine induces
repair-related premutagenic lesions and a novel form of
DNA hypomethylation that may relate to tumor promotion
in the methyl-deficient model of hepatocarcinogenesis.
Introduction
Uracil, a normal base in RNA, can appear in double-stranded
DNA by two distinct mechanisms. First, direct misincorpora*Abbreviations: UDG, uracil-DNA-glycosylase; AP, apyrimidinic.
© Oxford University Press
tion of dUTP in place of dTTP by DNA polymerase can occur
during DNA replication and repair, resulting in a U:A base
pair. Second, uracil can arise in DNA as a consequence of
cytosine deamination, resulting in a U:G mismatched base
pair. Regardless of the source, all uracil bases in DNA are
removed by an efficient and highly specific base excision DNA
repair enzyme, uracil-DNA-glycolsylase (UDG*) (1). UDG
recognizes the conformational change in DNA resulting from
the abnormal presence of uracil and binds to DNA such that
the damaged base is flipped out from the major groove into
the active site of the enzyme (2). The extrahelical uracil is
then excised and released from the DNA sugar–phosphate
backbone (3). Subsequently an AP endonuclease cleaves the
resulting apyrimidinic (AP) site, deoxyribophosphodiesterase
removes the 59-phosphate group, a DNA polymerase inserts
the correct nucleotide and DNA ligase completes the repair
process. Recent evidence suggests that UDG excises uracil
from U:G mispairs more efficiently than from U:A base pairs
(1,3), suggesting that repair of cytosine deamination may
exceed that of uracil misincorporation. Supporting this possibility is recent evidence that thymine-DNA glycosylase activity
acts in concert with UDG in excising uracil at U:G but not
U:A sites (4).
Over the last decade clinical and epidemiological evidence
has accumulated to indicate that inadequate dietary folate may
predispose to increased risk of certain types of human cancer
(5). A role for localized folate deficiency in the etiology of
human epithelial cell cancer has been indirectly implied by
the significant reversal of premalignant dysplasia in cervical
(6), bronchial (7) and colonic (8) epithelial cells with folate
supplementation. In recent epidemiological studies low folate
status has been associated with increased risk of colorectal
adenoma (9), rectal and colon cancer (10,11), cervical dysplasia, human papillomavirus infection (12,13) and liver and
esophageal tumors (14,15). In a recent US study increased
risk of colorectal cancer was associated with alcohol and low
methionine, low folate diets (11). In developing countries
epidemiological studies have linked monocereal diets (low in
methionine, choline and folate) to primary hepatoma (14) and
esophageal cancer (15). Marginal folate status has also been
associated with DNA lesions, including increased somatic
mutations induced by chemotherapy (16) and increased frequency of micronucleated erythroblasts (17). Two recent studies, using different methodologies, have confirmed the
inappropriate presence of uracil in lymphocyte DNA of folatedeficient humans (18,19). Further, Blount et al. have shown
that both uracil in DNA and micronucleus frequency in
splenectomized individuals can be reduced with folate
supplementation (19).
Previous experimental studies of rodent methyl deficiency
in vivo have documented increased genome-wide and genespecific cytosine hypomethylation (20–23) as well as increased
DNA methyltransferase activity, DNA strand breaks and apoptotic cell death (24,25). An increase in the ratio dUTP:dTTP
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I.P.Pogribny et al.
has recently been confirmed in liver of folate/methyl-deficient
rats (25) and has been postulated to account for the presence
of uracil in DNA (26–28). In vitro studies of folate deprivation
have demonstrated increased sensitivity to DNA damaging
agents associated with reduced DNA repair capacity (29)
and increased mutagenesis (30). Hepatocellular carcinoma in
rodents is inducible by a variety of methyl-deficient diet
formulations which may vary in tumor latency and frequency
as well as in preneoplastic histopathology (31–33). It is well
established that chronic deficiencies in the methyl donors
choline and methionine will increase the folate requirement
for endogenous resynthesis of methionine from homocysteine
(28) and reduce total folate content in the liver, despite
adequate folate in the diet (34,35). Because of the metabolic
interdependence of the lipotropic nutrients (choline, methionine, B12 and folic acid), it is likely that the different methyldeficient diet formulations promote hepatocarcinogenesis by
similar biochemical aberrations that may vary in severity and
onset. In the present study we have omitted folic acid from
the standard low methionine, choline-devoid methyl-deficient
diet in order to increase the severity of methyl deficiency, as
demonstrated previously (36–38). We show that chronic folate/
methyl deficiency in vivo leads to the inappropriate presence
of uracil in DNA, associated with increased genomic instability
as evidenced by repair-related AP sites and DNA strand breaks.
Loss of the thymine methyl group associated with uracil
substitution creates a hypomethylated site that can alter DNA–
protein interactions and interfere with normal gene expression
(39–41). These DNA lesions may interact to promote mechanisms of hepatocarcinogenesis in this nutritional model.
Materials and methods
Animals and diets
Male F344 weanling rats (50–60 g) were randomly allocated to receive either
a basal diet low in methionine (0.18%) without added folic acid or choline
(folate/methyl-deficient) or the basal diet supplemented with 0.4% methionine,
0.3% choline and 2 mg/kg folic acid (control). Folic acid was omitted from
the low methionine, choline-devoid diet as a means to enhance the severity
of the methyl group deficiency. The semi-purified diets were stored at 4°C
and replaced biweekly. Body weights and food consumption were recorded
weekly. No significant differences in body weights between diet groups were
recorded. Four rats/group were killed by CO2 inhalation at 3, 9, 24 and 36
weeks after diet initiation. The livers were excised and immediately frozen in
liquid nitrogen for subsequent DNA extraction. Genomic DNA was isolated
by digestion with proteinase K, phenol/chloroform extraction and ethanol
precipitation as described previously (42).
Immunohistochemistry
Formalin-fixed, paraffin-embedded liver sections were processed for immunohistochemical demonstration of GST-P by the biotin/extraavidin/peroxidase
detection system (Sigma Chemical Co., St Louis, MO). Primary antibodies
(rabbit polyclonal anti-human GST-P; Dako Corp., Carpinteria, CA) were
used at 1:1200 dilution. Non-specific staining was blocked with normal goat
serum (Sigma). Liver sections were incubated in biotinylated goat anti-rabbit
antibodies and extravidin-conjugated horseradish peroxidase. Staining was
developed with diaminobenzidine substrate and sections were counterstained
with hematoxylin.
Quantification of enzyme-induced DNA strand breaks in genomic DNA
A modification of the random oligonucleotide-primed synthesis (ROPS) assay
was used to detect the low frequency DNA strand breaks induced by
pretreatment with UDG and/or exonuclease III (Exo III) and has been described
previously in detail (43). The assay is based on the ability of Klenow fragment
polymerase to initiate ROPS from the reannealed 39-OH ends of singlestranded DNA. Briefly, after UDG and/or Exo III treatment, DNA containing
39-OH breaks is separated into single-strand fragments by heat denaturation
and subsequently reassociated by cooling. The resulting random reassociation
of DNA strands consists primarily of single-stranded DNA fragments primed
by their own tails or by other DNA fragments. These fragments serve as
random primers and the excess of DNA serves as template for the Klenow
2072
fragment polymerase. The incorporation of [32P]dCTP under strictly defined
conditions of time, temperature and precursor concentration is directly proportional to the number of enzyme-induced 39-OH breaks (fragment primers).
The results are expressed as the percent difference in [32P]dCTP incorporation
with and without enzyme treatment relative to control values.
Excision of uracil from DNA by UDG and cleavage of AP sites by Exo III
Uracil in DNA was excised by incubating 5 µg purified DNA in 50 µl TE
buffer for 2 h at 37°C with 5 U UDG (Epicentre Technologies, Madison, WI).
The AP sites generated by uracil excision were converted to DNA strand
breaks by subsequent treatment with Escherichia coli Exo III (New England
Biolabs). Exo III accounts for .80% of AP endonuclease activity in E.coli
and acts by hydrolyzing the phosphodiester bond 59 of an abasic site in DNA
(44). Briefly, the DNA was diluted with Exo III buffer (66 mM Tris–HCl,
0.66 mM MgCl2, 10 mM DTT, pH 8.0) and exposed to 2.5 U Exo III for 15
min at 25°C. The reaction was terminated by heat inactivation at 100°C for
10 min. Uracil in DNA was expressed as the percent increase in DNA strand
breaks in Exo III-treated DNA after exposure to UDG and is compared with
similarly treated DNA from control-fed rats. The relative level of AP sites in
DNA was expressed as the percent increase in Exo III-induced DNA strand
breaks without UDG treatment.
Comparative RT-PCR analysis of apurinic/apyrimidinic endonuclease mRNA
levels
Semi-quantitative or comparative RT-PCR analysis was used to measure the
relative levels of AP endonuclease mRNA in cell extracts from control and
folate/methyl-deficient rats (45). Briefly, total mRNA was extracted from liver
tissue utilizing the Quick-Prep Micro mRNA purification kit (Pharmacia
Biotech Inc., Piscataway, NJ). Exactly 0.5 µg mRNA were reverse transcribed
using the First Strand Synthesis kit (Pharmacia Biotech Inc.) according to the
manufacturer’s instructions. From each reaction tube 5 µl cDNA product were
PCR amplified using primers for AP endonuclease (59-CTGGACTTACATGATGAATGCCC-39 and 59-GAAGAGATAACGCACTGGTCTCCT-39). The
PCR reaction of consisted of 25 cycles of amplification (94°C for 30 s, 60°C
for 60 s and 72°C for 90 s). The PCR conditions were optimized to ensure
that the reaction was in the linear phase of amplification. As an internal
control for RT-PCR, equivalent amounts of cDNA were amplified by PCR
using glyceraldehyde 3-phosphate dehydrogenase primers. A parallel reaction
without addition of reverse transcriptase was used as a negative control for each
sample. Following amplification, 10 µl PCR products were electrophoresed on
2% agarose gels containing 0.5 mg/ml ethidium bromide in Tris–borate/EDTA
buffer and photographed under UV light.
Quantitative UDG PCR for detection of uracil within the p53 gene
Quantitative PCR was used to detect the relative level of DNA strand breaks
induced after UDG treatment within the p53 gene in DNA from folate/methyldeficient and control rats. This technique is based on the ability of strand
breaks to block the progression of Taq polymerase during PCR amplification.
Under optimized conditions the decrease in radiolabeled PCR product amplified
over a defined interval will be proportional to the level of enzyme-induced
DNA strand breaks present within the gene and is expressed as a percentage
of control samples (30). High molecular weight DNA was initially digested
with 4 U/µg SalI restriction enzyme (New England Biolabs, Beverly, MA)
for 12 h at 37°C to increase accessibility of the DNA to primer annealing.
Two micrograms of DNA were treated with UDG as described above. Intron
and flanking 20mer primer sequences for exons 5–8 of the rat p53 gene
have been previously published (46) and were synthesized by Genosys
Biotechnologies Inc. (Woodlands, TX). The PCR reaction conditions have
been previously described in detail and were optimized to ensure that the
reaction was in the linear phase of amplification (24). Results are expressed
as the percent change in 32P-labeled PCR product recovery relative to
control values.
Statistics
Significant differences between mean values were established using Student’s
t-test and Sigmastat software.
Results
Glutathione S-transferase P (GST-P)-positive hepatocellular
foci
After 9 weeks on the folate/methyl-deficient diet livers from
the deficient rats were grossly enlarged and markedly pale,
with a smooth capsular surface. The early increase in liver wt/
g body wt has been previously shown to be due to massive
lipid accumulation, the hallmark of ‘lipotropic’ methyl-
Consequence of uracil in preneoplastic DNA from folate/methyl-deficient rats
Figure 1. Representative example of immunohistochemical staining for
GST-P1 foci in liver sections after 24 weeks of folate/methyl deficiency
with hematoxylin counterstain. Note the GST1 focus associated with lobular
hyperplasia and lipid accumulation.
deficient diets (36,47). After 24 and 36 weeks of deficiency
the enlarged livers consisted of confluent parenchymal nodules
ranging in diameter from 0.5 to 2 mm. Microscopically,
prominent GST-P1 putative preneoplastic foci were present
after 24 weeks, as shown in Figure 1. These enzyme-altered
foci are thought to reflect an adaptive response to chronic
biochemical stress and a small percentage may evolve into
permanent premalignant lesions (48,49).
Relative levels of uracil in DNA, AP sites and DNA strand
breaks in methyl-deficient and control liver
The presence of uracil in hepatic DNA from methyl-deficient
and control rats was determined by quantifying the relative
number of enzyme-induced DNA strand breaks after Exo III/
UDG treatment as detected by the ROPS strand break assay.
In Figure 2A sites of UDG-mediated uracil excision were
increased 4-fold relative to the control after only 3 weeks
exposure to the deficient diet and remained significantly
elevated over the 36 week feeding period (P , 0.01). As
shown in Figure 2B, a significant increase in AP sites as
detected by Exo III treatment (P , 0.01) paralleled the increase
in uracil and similarly remained elevated over the 36 week
period. In contrast, accumulation of genome-wide DNA strand
breaks was incremental and progressive, as shown in Figure
2C, suggesting that an additional mechanism of DNA strand
breakage may be operative with chronic methyl deficiency.
AP endonuclease mRNA levels
Equivalent amounts of template mRNA from each sample
were reverse transcribed into cDNA strands which were
Figure 2. The effect of folate/methyl deficiency on genomic DNA breaks
was assessed by the ROPS assay in which the extent of [32P]dCTP
incorporation is directly proportional to the number of 39-OH breaks present
in the DNA. Bar heights are the means 6 SEM from 4 rats/group. Asterisks
indicate statistical difference from control (P , 0.01). (A) The relative level
of UDG/Exo III-induced breaks in DNA from rats fed deficient diet for 3,
9, 24 and 36 weeks is compared with the level of breaks from enzymetreated DNA from control rats. The percent increase in breaks after UDG/
Exo III treatment was calculated as: [(UDG/Exo III breaks – Exo III
breaks)/Exo III breaks]3100. (B) The relative level of Exo III-induced
breaks measured in DNA from control and deficient rats measured using the
ROPS assay. (C) The relative level of DNA breaks in untreated DNA from
control and deficient rats.
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I.P.Pogribny et al.
Figure 3. Ethidium bromide stained gel of comparative RT-PCR analysis of
AP endonuclease mRNA in cell extracts from control rat liver and after 9,
24 and 36 weeks of folate/methyl deficiency (n 5 2/group). The molecular
weight markers (MWM) indicate the expected molecular weight of AP
endonuclease. Note increased intensity of bands after 9 weeks of deficiency
relative to control.
subsequently amplified by PCR with primers specific for AP
endonuclease. The extent of PCR product amplification from
control and methyl-deficient samples is proportional to the
relative level of AP endonuclease mRNA present in the original
sample. In Figure 3 the PCR products amplified from cDNA
derived from control and deficient rats after 9, 24 and 36
weeks on the deficient diet have been photographed after
agarose gel electrophoresis. The increased intensity of the PCR
product bands amplified from hepatic cDNA derived from the
folate/methyl-deficient rats confirms that the steady-state level
of AP endonuclease mRNA is significantly increased in these
livers and parallels the increased presence of uracil and AP
sites in DNA.
Presence of uracil within the p53 gene
Based on previous observations of p53 gene-specific DNA
strand breaks and cytosine hypomethylation with folate/methyl
deficiency (23,24), we examined this sequence for the presence
of uracil using pretreatment with UDG followed by quantitative
PCR. Hepatic DNA from control and deficient rats was treated
with UDG to create AP sites in sequences containing unrepaired
uracil. The resulting AP sites were subsequently converted to
DNA strand breaks by E.coli Exo III treatment. The quantitative
recovery of 32P-labeled PCR product is proportional to the
number of enzyme-induced DNA strand breaks at sites of
uracil in DNA. As shown in Figure 4, a significant decrease
in quantitative recovery of PCR product was found with exons
5–8 of the p53 gene amplified from hepatic DNA from deficient
rats (P , 0.01), confirming the presence of uracil within this
gene sequence.
Discussion
The methyl group at C5 of uracil is the unique and distinguishing feature between the four canonical bases present in
DNA and those in RNA. Uracil is a normal base in RNA and
appears as 5-methyluracil (or thymine) in DNA. Whereas
cytosine in DNA is methylated in situ by a DNA methyltransferase after DNA synthesis, uracil is methylated in the cytoplasm by thymidylate synthetase before DNA synthesis and is
incorporated into DNA as a methylated base. The nomenclature
2074
Figure 4. The quantitative recovery of radiolabeled PCR product amplified
from exons 5–8 of the p53 gene after pretreatment of DNA template with
UDG of control rats and rats fed the folate/methyl-deficient diet for 36
weeks is compared with quantitative recovery of PCR product amplified
from non-treated DNA template (n 5 4/group). PCR product recovery is
inversely proportional to the level of uracil (UDG-induced breaks) within
the p53 gene sequence. PCR product recovery from the deficient rats was
significantly less than that from controls (asterisk, P , 0.01).
preference for ‘thymine’ as the unique base in DNA, rather
than ‘5-methyluracil’ has obscured the role of thymidine as
the second methylated base in DNA in addition to the wellstudied 5-methylcytosine. Recently the critical importance of
the thymine methyl group in maintaining normal base stacking
interactions for proper DNA conformation (50,51) and also for
sequence-specific DNA–protein binding has received increased
research attention (39,41). The thymine methyl group projects
into the major groove of the DNA where it has been shown
to alter sequence-specific binding of transcription factors such
as the cAMP-responsive element (39), the AP1 binding site
(TPA-responsive element) (40) and E.coli RNA polymerase
and lacI repressor (41). Thus uracil substitution for thymine
may be a genetically significant form of DNA hypomethylation
that can alter normal gene expression.
The two enzymes involved in preventing uracil accumulation
in active DNA are dUTP pyrophosphatase (dUTPase), which
hydrolyzes dUTP to dUMP and PPi, and UDG, which cleaves
the glycosylic bond between uracil and the DNA sugar–
phosphate backbone, releasing uracil. Cytoplasmic dUTPase
is a cell cycle-regulated enzyme that acts to maintain a low
dUTP:dTTP ratio in actively proliferating cells (52,53). Since
DNA polymerases do not distinguish between dUTP and dTTP,
a low dUTP:dTTP ratio is essential to avoid inappropriate
uracil incorporation during DNA replication/repair. However,
because the activity of dUTPase is generally low in nonproliferating cells (52), the potential for uracil (mis)incorporation during DNA repair synthesis may be greater in quiescent
than in proliferating cells (54). An essential early response to
proliferative stimuli is a sharp increase in UDG activity (55,56),
which would be consistent with accumulation of uracil in
resting cells. Further, the peak of UDG activity before mid S
phase underscores the importance of uracil excision and repair
before entry into S phase and DNA synthesis.
Misincorporation of uracil for thymine per se is not a
Consequence of uracil in preneoplastic DNA from folate/methyl-deficient rats
premutagenic lesion because the polymerase will insert the
correct adenine base opposite either thymine or uracil, whereas
cytosine deamination to uracil can result in a C→T transition
mutation if not repaired before DNA replication. Thus the
negative consequences of uracil in DNA are indirect and
cumulative and may involve a threshold effect when the
capacity for uracil excision is exceeded. The enzymatic repair
of uracil involves extrahelical flipping of uracil into the active
site of UDG, which creates an AP site, followed by an
endonuclease-mediated DNA strand breakage (57). Interestingly, excessive accumulation of AP sites in DNA induces a
feedback inhibition of UDG, presumably to preserve minimum
essential DNA helix stability (58). This has led to the suggestion
that UDG activity may be regulated by the ratio of uracil to
AP sites in DNA in order to preserve helical stability (50).
Helical stability would be further compromised by the 4-fold
increase in liver DNA methyltransferase activity induced by
folate/methyl deficiency (24), since the methyltransferase,
similar to UDG, involves enzyme-assisted extrahelical nucleotide flipping (2).
The presence of uracil in leukocytes and bone marrow DNA
from folate-deficient humans has been previously documented
using different methodologies for detection (19,59,60) and
has been associated with micronuclei formation (61,62) and
induction of folate-sensitive fragile sites (63). In the present
study we have found detectable uracil in hepatic DNA as early
as 3 weeks after feeding a folate/methyl-deficient diet to
F344 rats. In previous studies we demonstrated that the ratio
dUTP:dTTP in liver is significantly elevated after 3 and 9
weeks of folate/methyl deficiency (25,64) and that it is also
increased in folate-deprived CHO cells in vitro (30). The
increase in the ratio dUTP:dTTP, stemming from reduced
folate-dependent thymidylate synthesis, has been postulated to
promote uracil incorporation, DNA fragmentation, fragile sites,
cell transformation and cell death (27,50,65–67).
In this report we extend the evidence that excessive uracil
in DNA has a deleterious effect on the structural integrity of
DNA, as evidenced by the parallel increases in uracil, AP sites
and DNA strand breaks with increasing duration of folate/
methyl deficiency. The significant elevation in AP endonuclease
expression is consistent with an increased level of AP sites.
Although evidence for the existence of uracil within the p53
gene was also obtained, it is not possible to determine from
these data whether this is a gene-specific event or a reflection
of genome-wide prevalence. Based on these and previous
observations of cytosine hypomethylation, we hypothesize that
both uracil misincorporation and deamination of cytosine
combine to exceed the uracil base excision repair capacity and
promote the presence and consequences of uracil in DNA in
the preneoplastic liver of methyl-deficient rats. The substitution
of uracil for thymine in DNA constitutes a novel form of
DNA hypomethylation that may affect expression of cancer
promoting genes.
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Received on May 1, 1997; revised on July 8, 1997; accepted on July 30, 1997