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Carcinogenesis |
Medical Research Council Toxicology Unit, Centre for Mechanisms of Human Toxicity [Y. G., P. B. F.] and Department of Chemistry [G. D. D. J.], University of Leicester, Leicester LE1 9HN, United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The role of PTE adducts in carcinogenesis is unknown. Mutation resulting from PTE formation has not been fully studied, but such adducts may influence cellular function by altering the binding of proteins to DNA (7) . For certain alkylating carcinogens, the resulting PTE adducts are chemically stable in DNA, and their relative abundance has been reported in various studies (8 , 9) . The proportion of total alkylation products manifest as PTE adducts in salmon sperm DNA treated with ethylnitrosourea is 55.3%, whereas for the less carcinogenic agent ethylmethanesulfonate, PTE adducts constitute 12.0% of total alkylation products (9) . Furthermore, the extent of PTE formation tends to parallel that of other oxygen alkylation products such as on the O6-alkylated adduct of guanine or the O4 -alkylated adduct of thymine, which are known to be highly mutagenic. O6-Ethylguanine represents 9% of DNA alkylation products for ethylnitrosourea and 2% of DNA alkylation products for ethylmethanesulphonate (9) . Thus, PTEs might serve as valid markers for known mutagenic lesions in DNA.
In Escherichia coli, methyl PTE can be removed by the native form of the O6-methylguanine-DNA methyl transferase (Ada protein; Ref. 10 ). This repair mechanism is inducible (11) and specific for the S-isomer of methyl PTE (10 , 12 , 13) . A related mechanism has also been reported in the eukaryote Aspergillus nidulans (14) , but a mechanism for repair of alkyl PTE in higher eukaryotic systems has not yet been demonstrated. Studies using rodents (15, 16, 17) or human fibroblast cell lines (18) exposed to alkylating agents have demonstrated the long biological half-lives of methyl PTE and ethyl PTE, which exceed those of other known DNA alkylation products. Thus, PTE lesions may serve as ideal biomarkers with which to assess cumulative genotoxic exposure.
One of the reasons that so little attention has been paid to the detection of carcinogen-induced PTEs in DNA has been the lack of suitable analytical methods. Consequently, the extent of PTE formation by most carcinogens is largely unknown. Early efforts to quantify total PTE in DNA involved analysis of the alkali-induced strand break at the sites of PTE lesions (3 , 19 , 20) , but this is a relatively nonspecific approach presumably confounded by the presence of other alkali-labile lesions in the DNA. Recently, a different approach exploiting the transalkylation of PTE to a stronger nucleophile has been reported in attempts to identify the PTE-forming species (21) , but this method has not been applied to in vivo studies.
The most frequently used methods for PTE adduct detection have exploited the inability of known nucleases to cleave the PTE internucleotide linkages in DNA (22 , 23) . Consequently, digestion of PTE-containing DNA with nucleases, plus a phosphatase, yields PTE-dinucleoside phosphates (2'-deoxynucleoside 3'-[alkyl]phosphate-2'-deoxynucleosides), with the remaining DNA being recovered as mononucleosides. Using radiolabeled alkylating agents and radioactivity detection of eluates from ion-exchange chromatography of the enzymatic DNA digests, the alkyl PTE of thymidylyl (3'-5') thymidine, dTp(alkyl)dT, has been detected in vitro and in vivo in animal experiments (9 , 16 , 17 , 24 , 25) . However, these methods are not appropriate for human studies and are unsuitable for the detection of PTEs that may be produced endogenously.
The possibility of detecting PTE in human DNA in vivo
has recently been advanced by developments in 32P
postlabeling methods (26
, 27) . The PTE-dNpdNs obtained
from the nuclease plus phosphatase-mediated digestion of PTE-containing
DNA (see above) are not substrates for phosphorylation by T4 PNK
because one of the substrate requirements of this enzyme is the
presence of a negative charge on the phosphate moiety closest to the 5'
end (28
, 29)
. Therefore, before the labeling step, these
species are converted into either dNpdNs or 3'-phosphate alkylated
mononucleosides [dNp(R)] by alkali hydrolysis. Both the dNpdN and
dNp(R) species produced are ready substrates for 5'
32P end labeling by incubation with T4 PNK and
[
-32P]ATP. Up to 16 labeled dinucleotides
(32pdNpdN) and 4 labeled dNp(R)
[32pdNp(R)] species may be obtained, depending
on the nature (and frequency) of the neighboring nucleosides, with the
relatively low yield of 32pdNp(R) species,
compared with 32pdNpdN species, limiting their
detection in DNA (26)
. The resulting complex mixture of
labeled compounds has previously been analyzed by a combination of TLC
and HPLC (26)
, but this two-step analysis is inappropriate
for routine analysis. The present study describes a nuclease plus
phosphatase-based 32P postlabeling method for the
detection of total alkyl PTE in DNA with analysis and quantitation of
the labeled dinucleotides by PAGE (Fig. 1)
. In addition, this method allows for identification of the nucleosides
located 5' to the alkylated phosphate by NP1-mediated digestion of the
gel-isolated 32pdNpdN species and analysis of the
released labeled nucleotides (32pdN) by HPLC with
radioactivity detection (Fig. 1)
. The method has been validated using a
prepared methyl PTE-containing oligonucleotide possessing a single PTE
adduct at a defined location and has been applied to the analysis of CT
DNA treated in vitro with DMS or DES and to the analysis of
liver DNA from mice treated in vivo with NDEA.
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MATERIALS AND METHODS |
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-32P]ATP (Redivue; >3000 Ci/mmol) was from
Amersham International (Little Chalfont, United Kingdom). All others
reagents and solvents of ultra pure grade were purchased from either
National Diagnostics (Hull, United Kingdom) or Life Technologies, Inc.
(Paisley, United Kingdom). The dNpdNs, dTp(Me)dT, and methyl
PTE-containing oligonucleotide
[(dT)3dTp(Me)(dT)7] were
prepared by the Protein and Nucleic Acid Chemistry Laboratory (CMHT,
University of Leicester, Leicester, United Kingdom). Liver DNA from
SWR, BALB/c, and C57BL/6J mice treated in vivo with a single
i.p. dose of NDEA (90 mg/kg) was provided by Drs. R. Singh and V.
Oreffo (Medical Research Council Toxicology Unit).
Enzymes and Buffers
DNase I (type II from bovine pancreas) and SVPD (type IV from
Crotalus atrox) were purchased from Sigma Chemical Co. Ltd.
NP1 was purchased from Pharmacia Biotech. Shrimp alkaline phosphatase,
T4 PNK, and kinase buffer were purchased from Amersham International.
Alkylation of CT DNA
CT DNA (1 mg/ml, 
3 mM DNA-P) was methylated or
ethylated by treatment with DMS or DES, respectively, (1–10
mM) in sodium phosphate buffer (0.5 M, pH 6.0)
at room temperature for 8 h (8)
and then precipitated
by the addition of 0.1 volume of sodium acetate (3 M) and
0.8 volume of 2-propanol. The DNA pellet was washed twice with 70%
ethanol, evaporated to dryness using a DNA centrifugal evaporator (DNA
110; Savant, Farmingdale, NY), and then dissolved in water to a
concentration of 1 mg/ml as determined by UV absorbance spectrometry.
Digestion
DNA, the methyl PTE-containing oligonucleotide, and the methyl
PTE dinucleoside phosphate were all "digested" using a modification
of the procedures described by Weinfield et al.
(30)
and Saris et al. (26)
.
Samples of DNA (10 µg), methyl PTE-containing oligonucleotide (32
pmol), or methyl PTE dinucleoside phosphate (8 pmol) were incubated
overnight at 37°C with DNase I (0.4 unit), SVPD (0.02 unit), NP1 (0.5
unit), and shrimp alkaline phosphatase (0.4 unit) in 30 µl of
digestion buffer [10 mM Tris-HCl (pH 7.4), 1
mM EDTA, 4 mM
MgCl2, and 2 mM
ZnCl2]. The enzymes were then precipitated by
the addition of 3 volumes of ice-cold ethanol and removed by
centrifugation at 4°C (17,000 x g, 15
min). The supernatant was evaporated, and the resulting residues were
dissolved in water (100 µl), heated at 100°C for 10 min to
inactivate residual nuclease and phosphatase activity, and then stored
at -20°C.
Alkali Hydrolysis of PTE
Hydrolysis with ammonia of the digested PTE-containing DNA,
oligonucleotide, or dinucleoside phosphate was carried out by using the
procedure described by Saris et al. (26)
. An
equal volume of aqueous ammonia (25%) was added to an aliquot (50
µl) of digest solution, and the reaction was incubated in a tightly
capped vial at 70°C for 24 h. The reaction mixture was
evaporated to dryness using a DNA centrifugal evaporator, and the
residue was dissolved in an appropriate volume of water (10–50 µl)
and then stored at -20°C.
32P Labeling
Each phosphorylation reaction mixture (10 µl) contained the
supplied kinase buffer (used 1x), 5 µl of the digested solution
(untreated or treated with alkali) or the PTE-containing
oligonucleotide or known amounts of the dinucleoside phosphate
standards, 5 µCi of [-32P]ATP (1 pmol),
T4 PNK (7.5 units), and 100 fmol of poly(dT)16
when used as a labeling reference. The samples were incubated at 37°C
for 1 h. When poly(dT)16 was used to consume
excess [-32P]ATP, the reaction was incubated
for an additional 30 min with 40 pmol of
poly(dT)16 plus T4 PNK (4 units). At the end of
the reaction, an equal volume of formamide loading buffer (90%
formamide, 0.02% bromphenol blue, and 0.02% xylene cyanol in 100
mM Tris, 90 mM boric acid, and 1 mM
EDTA; Ref. 31
) was added, and 5 µl of the mixture were
analyzed by PAGE.
PAGE of the 32P-labeled Compounds
Samples were loaded onto a 20% polyacrylamide/7 M urea
gel. The gel electrophoresis equipment used consisted of a power supply
(EPS 3500 XL; Pharmacia Biotech) together with a Model S2 Sequencing
Gel Electrophoresis System unit using 33.5 (width) x 41.5-cm (length) glass plates, 0.8-mm spacers, and a 32-tooth comb
(Life Technologies, Inc.). For standard conditions, electrophoresis was
carried out at 1100 V until the bromphenol blue marker migrated 11–12
cm (3–4 h). Radiolabeled products were visualized by autoradiography
on Kodak X-OMAT K film (30 x 24 cm) or by storage
PhosphorImager analysis (model 42SE; Molecular Dynamics, Sunnyvale, CA;
using ImageQuant software version 3.2). The level of dNpdN (fmol) in a
sample was determined as follows: (fmol of dNpdN used in the standard
or mixture of standards) x (integrated volume for
32pdNpdN obtained from the samples)/(integrated
volume for 32pdNpdN obtained from the standard or
mixture of standards). When poly(dT)16 was used
as a labeling reference, the obtained value was multiplied by the
following correcting factor: [integrated volume for labeled
poly(dT)16 obtained from the
standard(s)]/[integrated volume of labeled
poly(dT)16 obtained from the samples].
Analysis of the 5'-labeled Nucleotides in the
32pdNpdN Species
The radioactive fragments of gel containing the
32pdNpdN species were excised from the gel and
then soaked overnight at room temperature in 1 ml of water. After
removing the water, the gel fragments were rinsed with an additional
0.5 ml of water, and the washes were combined. Before NP1 digestion,
the combined washes were desalted by lowering the pH to 4 (by adding an
appropriate volume of 50 mM HCl solution) and then applying
the mixture to a Water Sep-Pak C18 light
cartridge previously conditioned in a 50 mM ammonium
formate buffer (pH 4). The cartridge was washed with 10 ml of the same
buffer, and 32pdNpdN species eluted with 0.8 ml
of 50% v/v methanol/water mixture. The obtained fraction was taken to
dryness and resuspended in 50 µl of NP1 buffer (10 mM
sodium acetate and 1 mM ZnCl2). After
adding NP1 (0.6 unit), the reaction mixture was incubated for 1 h
at 37°C and subjected to HPLC with all four 5'-mononucleotides as UV
markers. HPLC was performed using a Varian Star 9012 pump system
(Varian United Kingdom Ltd., Surrey, United Kingdom) connected with a
UV-Vis detector (Varian Star 9050) and a radioactivity detector
(ß-ram; LabLogic, Sheffield, United Kingdom). The data were analyzed
by Laura software (LabLogic). Absorbance of the eluate was monitored at
254 nm. Analyses were performed at room temperature using a
reverse-phase C18 column (apex, 5 µm;
4.6 x 250 mm; Jones Chromatography Ltd.) fitted with a
C18 guard column (Techsphere; 5 µm; 3 x 10 mm; HPLC Technology Co. Ltd., Macclesfield, United Kingdom).
The HPLC gradient conditions were as follows: solvent A, 50
mM ammonium acetate (pH 4.5); solvent B, methanol/water
(80:20, v/v); gradient: 0 min, 0% B; 5 min, 0% B; 20 min, 20% B; and
25 min, 0% B at a flow rate of 1 ml/min.
Analysis of Normal Mononucleosides in DNA Digests
Aliquots (10 µl) of digested DNA samples and an equimolar mixture of
four 2'-deoxynucleoside standards were subjected to HPLC using a Waters
2690 pump connected with a Waters 486 UV-Vis detector (Waters, Milford,
MA). The data analysis was performed using Millennium software, a
Microsoft Windows package (Waters). Absorbance of the eluate was
monitored at 260 nm. Analyses were performed at room temperature using
a reverse-phase C18 column (Hypersil BDS C18; 3
µm; 2.1 x 100 mm; Shandon HPLC) fitted with a
C18 guard column (Techsphere; 5 µm; 3 x 10 mm; HPLC Technology). Isocratic elution was conducted using
0.1 M triethylammonium acetate (pH 5.0) and 4% methanol at
a flow rate of 0.2 ml/min.
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RESULTS |
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, a methyl PTE dinucleoside phosphate [dTp(Me)dT] and an
oligonucleotide containing a single methyl PTE
[(dT)3dTp(Me)(dT)7] were
examined. The protocols for the 32P labeling
stage and separation of the resulting labeled compounds by PAGE were
adapted from protocols described by Weinfeld et al.
(30
, 32)
for the analysis of oxidative and radiogenic DNA
damage. The autoradiogram in Fig. 2
shows the labeled compounds obtained after various treatments of
(dT)3dTp(Me)(dT)7 and
dTp(Me)dT. Each sample contained a limiting amount of
poly(dT)16 as a labeling reference. In parallel,
the dinucleoside phosphate dTpdT was labeled under identical
conditions. Alkali hydrolysis of the enzyme-digested samples was
carried out using aqueous ammonia according the protocol of Saris
et al. (26)
. This protocol was judged
appropriate because labeling of the digested and alkali-treated methyl
PTE-containing oligonucleotide and the digested alkali-treated
PTE-dinucleoside phosphate yielded the anticipated labeled dinucleotide
(32pdTpdT). This species was also detectable in a
small amount in samples that have been labeled without the alkali
treatment. This is presumably the result of some minor hydrolysis of
the bonds of the phosphate esters during the heating of the digest
sample to inactivate residual nuclease and phosphatase activity (see
"Materials and Methods").
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60%.
32P Labeling of All of the Possible
Dinucleoside Phosphates
The autoradiogram in Fig. 3A
shows the labeled compounds obtained on
32P labeling of all possible dinucleoside
phosphates expected after digestion and alkali hydrolysis of alkylated
DNA. Sixteen authentic dinucleoside phosphate standards were analyzed
individually and in an equimolar mixture. For these experiments,
residual [-32P]ATP was consumed using a
large excess of poly(dT)16 (see "Materials and
Methods"). Results obtained from the equimolar mixture of standards
indicated that under the gel electrophoresis conditions used, the
32pdNpdN species are grouped into
five prominent bands, bands 1–5. Their relative intensity, as measured
by storage phosphor image analysis, was approximately 1:4:6:4:1. Bands
1 and 5 contain only the 32pdGpdG and
32pdCpdC species, respectively; bands 2, 3, and 4
consist of labeled dinucleotides containing the base sequences [GA,
GT, AG, TG], [CG, GC, AT, TA, TT, AA,] and [TC, CT, AC, CA],
respectively. For the individual dinucleoside phosphates, the molar
quantity of 32pdNpdN species in each band was
determined by multiplying the fraction of the total activity of the
lane found in each band by the molar quantity of
[-32P]ATP used in the reaction. The results
indicated a similar labeling efficiency for each dinucleoside
monophosphate of 50 ± 15%.
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shows the ratio of the 16 32pdNpdN
species:labeled poly(dT)16 observed when
0.39–100 fmol of the equimolar mixture of dNpdN standards were labeled
in the presence of a limiting amount of
poly(dT)16. At levels of the dNpdN standards
lower than 3.12 fmol (<0.2 fmol/dNpdN), the measurements obtained were
indistinguishable from those of control samples containing just water.
This background appears because of compounds present in the commercial
preparations of T4 PNK or [-32P]ATP. These
contaminants, once labeled, comigrate with the
32pdNpdN species on gel electrophoresis. From
these experiments, we judged the detection limit of this assay to be
5 fmol for the detection of the 16 dinucleoside phosphates.
Analysis of in Vitro Alkylated Modified DNA
The conditions established above were applied to the detection of PTE
DNA adducts formed by reacting an alkylating agent with DNA in
vitro.
In the first experiment, CT DNA was incubated with 10
mM DES. A control sample was an equal concentration of CT
DNA in the same buffer without DES. Before the labeling step, DNA
digests were either nontreated or treated with alkali. Residual
[-32P]ATP was consumed by an excess of
poly(dT)16. In parallel, an equimolar mixture of
dNpdN standards was labeled under identical conditions. Compared with
control DNA digests, autoradiograms obtained from DES-treated DNA
digests revealed the presence of five prominent bands corresponding to
32pdNpdN species (Fig. 4)
. These bands were far more intense in the alkali-treated digest,
indicating that the detected dinucleoside phosphates arise from
PTE-dinucleoside phosphates generated by digestion of DES-treated DNA.
By comparison with the mixture of standards labeled under identical
conditions (see "Materials and Methods"), the level of dinucleoside
phosphates was calculated as 200 fmol. The detectable bands in the
nonalkali-treated digest may be due to a partial PTE hydrolysis during
sample preparation (see above). The detected activity in the
alkali-treated control samples is at the detection limit of the assay.
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shows the resulting autoradiogram of the polyacrylamide gel
and demonstrates the appearance, with increasing doses of DMS and DES,
of the five characteristic bands indicating the formation of methyl-PTE
and ethyl-PTE, respectively. Levels of dinucleoside phosphates in each
sample were determined by comparison with the mixture of standards
labeled under identical conditions (see "Materials and Methods").
Fig. 5B
shows a clear dose response for the detected
dinucleoside phosphates as the concentration of the alkylating agent
used in the in vitro treatment was increased. The levels of
PTE adducts in DNA samples were calculated by taking into account the
amount of digested and alkali-treated DNA used for analysis and the
overall yield of the method (60%). The observed levels vary from
0.9–17.5 methyl PTE/104
DNA-P in the 0.1–10
mM DMS-modified samples and from 0.4–9.2 ethyl
PTE/104
DNA-P in the 0.5–10
mM DES-modified samples.
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. The levels of ethyl PTE adducts in liver DNA from the three strains
of treated mice were calculated as 25.3 ± 4.1
PTE/106 DNA-P.
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. In both cases, the major labeled eluted compounds correspond to one of
the expected four normal 5'-mononucleotides in DNA. The relative
amounts of each radiolabeled nucleotide were obtained from the observed
area peak ratios. The variation of this assay, based on the repeated
analysis of an equimolar mixture of the dinucleoside phosphate
standards (which were labeled, gel-isolated, and NP1-digested), was
found to be 
10% (data not shown). Table 1
shows the frequency of the nucleosides 5' to the PTE lesions obtained
from in vitro and in vivo studies compared with
the normal nucleoside content as determined by HPLC analysis of the
digested DNA samples (see "Materials and Methods"). Results
obtained from CT DNA treated in vitro with DMS or DES show
that the frequency of the four nucleosides 5' to the PTE adducts
reflected the normal nucleoside content. Results obtained for liver DNA
from mice treated in vivo with NDEA are different. The
frequency of the thymidine 5' to the PTE adducts was significantly
higher than the normal thymidine content, whereas the frequency of
2'-deoxyguanosine 5' to the PTE adducts was significantly less than the
normal 2'-deoxyguanosine content.
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DISCUSSION |
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For the digestion stage, we followed the protocol described by Weinfeld et al. (30) and Saris et al. (26) , which used NP1 in addition to SVPD for the digestion of DNA to nucleosides (33) . The two exonuclease enzymes have different specificities. SVPD interacts with the bases 3' to a phosphodiester linkage, and a missing base or certain modified bases at this position inhibit cleavage of the phosphodiester bond. On the other hand, NP1-mediated phosphodiester cleavage is inhibited by a missing base or certain modified bases immediately 5' to the phosphodiester linkage (34) . Results obtained in our laboratory have shown that the use of these two enzymes simultaneously for the digestion of alkylated DNA allows for the sole detection of PTE-derived dinucleoside phosphates, with other possible lesions such as alkylated bases and apurinic/apyrimidic sites being shown not to interfere (data not shown). The specificity of the method was confirmed by the fact that the band intensity of the dinucleoside phosphates corresponding to PTE adducts were strongly amplified in the alkali-treated samples. Our method, which is designed for the analysis of PTE-derived labeled dinucleotides, could be applicable to the detection of any carcinogen-induced PTE as long as the PTE-dinucleoside phosphates obtained after digestion can be hydrolyzed to yield the corresponding dinucleoside phosphates.
Quantitation of total PTE in DNA was estimated from the level of
PTE-derived dinucleoside phosphates detected. At this time, the
sensitivity of the method is limited by interfering compounds present
in the labeling reaction. The minimum detectable amount of dNpdN
species as an equimolar mixture was 5 fmol, allowing the detection
of 2–3 PTE/106 DNA-P when 0.5 µg of DNA is
used. The yield of the method was determined to be 60%, using a
site-specific methyl PTE oligonucleotide. However, despite a similar
labeling efficiency for the 16 dinucleoside phosphates, it is uncertain
whether the result obtained for the detection of a single PTE lesion
contained in a single homo-oligonucleotide can be extrapolated to the
circumstance of alkylated DNA because of the potential for
sequence-dependent differences in the yields of products on alkali
hydrolysis (35)
. Thus, levels of PTE adducts determined in
this study represent a semiquantitative approximation. However, the
results obtained in vitro showed that levels of methyl PTE
in DNA treated with DMS are, on average, twice that of the levels of
ethyl PTE in DNA treated with DES. This is in agreement with the
previous observation that methylating agents are more reactive than
ethylating agents to form PTE in DNA (36)
. Furthermore,
our values obtained from liver DNA of mice treated with NDEA in
vivo are in the same range (10–30
ethyl-PTE/106 DNA-P) as those obtained from liver
DNA of rats given NDEA in drinking water (where the total level of PTE
was measured by alkali-induced strand break analysis; Ref.
3
). In addition, our estimations of ethyl-PTE are, on
average, twice the levels of N-7 ethylguanine and
O6 ethylguanine DNA adducts that have
been determined by immunoslot-blot analysis of the same
samples.4
The latter observation is in broad agreement with results obtained from
a study of rats treated with ethylnitrosourea, where the total level of
ethyl PTE [extrapolated from dT(ethyl)pdT detected by HPLC] were
found to be three times higher than the level of N-7
ethylguanine in liver DNA (16)
. The levels of any PTE
adducts in liver DNA of nontreated mice were below the detection limit
of our assay; analogously, no base adducts were detected by
immunoslot-blot analysis of the same samples.4
An obvious drawback of this postlabeling method is that for every type of PTE generated, up to 16 labeled compounds can be produced, and these are not fully separated by PAGE. On the other hand, advantage can be taken of this situation through the use of NP1 to obtain the identity and frequency of the nucleosides located 5' to the alkylated phosphates. The outcome of this type of analysis carried out on CT DNA modified with DMS or DES indicates that the frequency of the four nucleosides 5' to PTE reflects the normal base content of CT DNA, suggesting that the reaction of the alkylating agents with internucleotidic phosphodiester groups within a double-strand DNA is random in vitro. However, analysis of liver DNA from three strains of mice treated in vivo with NDEA indicates that the frequency of the thymidine and the 2'-deoxyguanosine 5' to the ethyl PTE is significantly different from the corresponding normal nucleoside content. Because DES and NDEA give the same PTE products in DNA, the difference in the results in vitro and in vivo suggests (a) the nonrandom formation of ethyl PTE in vivo; and/or (b) sequence-specific ethyl PTE repair. With regard to (a), this could arise from sequence-dependent differences in the chemical reactivity of DES and metabolized NDEA toward phosphate groups in DNA. However, with regard to (b), despite the fact that PTE DNA repair has not yet been reported in the eukaryotic system, the results obtained in vivo could suggest the intervention of a sequence-specific ethyl PTE repair mechanism. Overall, the results obtained in this study are in agreement with the general acceptance that adduct distribution in the genome is not random (2 , 37) . In many cases, because of the structural organization of the genome, base-adduct formation has preferential sites of alkylation in DNA, some of which have been shown to be sequence dependent (38) . Furthermore, lesion repair has been shown to be base sequence dependent, varying in extent between individual nucleotide sites (39, 40, 41) , as well as dependent on the genomic localization of the lesion and the functional status of the gene (42, 43, 44) .
To date, there is no record of a compound that reacts with the phosphates of nucleic acids that is not a carcinogen. This observation prompts the question as to whether the level of such lesions can be related to cancer incidence. Because our assay for PTE involves the determination of the dinucleoside phosphates derived from PTE adducts (after digestion and alkali hydrolysis), it should have a wide applicability. One of the ultimate aims is to apply the assay to the analysis of human DNA from populations exposed to suspected environmental carcinogens. Further methodological development will focus on analysis of the carcinogen-containing species that are also released during the alkali hydrolysis. Our assay should permit a study of the role of the DNA sequence in the formation and removal of PTE. This type of information will be helpful in understanding the mechanisms of PTE adduct formation and their biological significance in vivo.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a Medical Research Council
Realizing Our Potential Awards grant (to P. B. F.). 
2 To whom requests for reprints should be
addressed, at Medical Research Council Toxicology Unit, Hodgkin
Building, University of Leicester, P. O. Box 138, Lancaster Road,
Leicester LE1 9HN, United Kingdom. Phone: 44-116-252-5619; Fax:
44-116-252-5616; E-mail: pbf1{at}le.ac.uk
3 The abbreviations used are: PTE,
phosphotriester; dNpdN, 2'-deoxynucleoside
3'-phosphate-2'-deoxynucleosides (dinucleoside phosphates); dNp(R),
2'-deoxynucleoside 3'-[alkyl]phosphate; dTp(Me)dT, 2'-thymidine
3'-[methyl]phosphate-2'-thymidine; CT, calf thymus; DMS,
dimethylsulfate; DES, diethylsulfate; NDEA, nitrosodiethylamine; T4
PNK, T4 polynucleotide kinase; SVPD, snake venom phosphodiesterase;
NP1, nuclease P1; HPLC, high-performance liquid chromatography; CMHT,
Centre for Mechanisms of Human Toxicity; poly(dT)16,
polydeoxythymidylic acid oligonucleotide; DNA-P,
DNA-phosphates.
4 V. Oreffo, R. Singh, K. J. Rich, D. E. G.
Shuker, P. Carthew, R. Cordero, J. van Delft, and P. B. Farmer. DNA
adducts in relation to lung tumour outcome are not markers of
susceptibility following a single dose treatment of SWR, BALB/c and
C57BL/6J mice with N-nitrosodiethylamine, submitted for
publication.
Received 5/24/99. Accepted 1/ 4/00.
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