Mutagenesis vol. 20 no. 4 pp. 305–310, 2005
Advance Access publication 14 June 2005
doi:10.1093/mutage/gei042
Wavelength dependent responses of primary human keratinocytes to
combined treatment with benzo[a]pyrene and UV light
Rebecca A.Crallan, Eileen Ingham1 and
Michael N.Routledge
Molecular Epidemiology Unit, Centre for Epidemiology and Biostatistics,
Leeds Institute for Genetics, Health and Therapeutics and 1The School of
Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK
The major risk factor for skin cancer is exposure to UV
radiation from sunlight, but other environmental exposures
may also play a role in combination with UV. We have
studied the effects of combined exposure of primary human
skin cells in vitro to UVA, UVB or UVC with benzo[a]
pyrene (BaP), an environmental carcinogen. Normal
human keratinocytes were exposed to 5 mM BaP for 24 h
followed by either 1 kJ/m2 UVA, 100 J/m2 UVB or 10 J/m2
UVC. Only BaP 1 UVA caused increased cell death. BaP
or UVA alone did not induce significant DNA damage as
measured by comet assay but combined exposure induced
35.1 6 6.0% tail DNA, compared with 9.7 6 1.3% tail DNA
in control cells. After including the Fapy-DNA glycosylase
enzyme incubation step to detect oxidized purines, % tail
DNA increased another 11.2 6 2.9%. Combined exposure
of BaP and UVB did not increase damage in the comet assay
without Fapy-DNA glycosylase, but in the presence of this
enzyme % tail DNA increased by 9.3 6 2.2%. BaP 1 UVB
also abrogated the UVB-induced cell cycle G2 arrest.
BaP 1 UVC had no effect on the keratinocytes compared
with each treatment alone. These results show a wavelengthdependent difference in the effects of combined exposure on
normal human keratinocytes. Both UVA and UVB damage
can be enhanced by BaP pre-exposure, although the effects
seen with UVA were greater. These findings are important
to understanding the role of UVA and UVB in skin carcinogenesis and may have implications for recommended sun
exposure limits, especially in polluted areas.
Introduction
Skin cancer is the most common form of cancer in the UK.
The vast majority (>90%) are non-melanoma skin cancers
(NMSCs), either basal or squamous cell carcinomas that arise
from keratinocytes in the epidermis of the skin. Over 60 000
new cases are diagnosed each year, an incidence that is
increasing (1,2) (http://www.cancerresearchuk.org/aboutcancer/
reducingyourrisk/sun_uv). Cumulative sunlight exposure has
been shown by numerous epidemiological and animal studies
to be the most important risk factor (3–5), but exposure to
pollution, cigarette smoke and occupational chemicals also
plays a significant role in the development of NMSCs (6–9).
UV radiation is the carcinogenic component of sunlight.
The sun emits UVA (320–400 nm), UVB (280–320 nm) and
UVC (<290 nm), as well as visible light. UVC is absorbed by
molecular oxygen (O2) in the atmosphere and most of the UVB
is absorbed by the ozone layer (O3); consequently, 95% of UV
reaching the earth’s surface is UVA. All wavelengths of UV
can induce DNA damage, although different spectra of lesions
are associated with different wavelengths. UVC and UVB are
very efficient at producing cyclobutane pyrimidine dimers
(CPDs) and (6-4)photoproducts (10). UVA is poorly absorbed
by DNA and induces much lower levels of CPDs, but generates
reactive oxygen species (ROS), resulting in oxidative DNA
damage (11–12). Despite these differences, the relative contribution of UVA versus UVB to NMSC development is still
not clear; although the majority of CPDs induced by sunlight
and sunlamps in cultured cells are caused by UVB radiation
(13) and GC!AT transitions are the most common mutations
found in NMSCs (14), UVA exposure alone can cause skin
cancer (15). Higher levels of UVA than UVB penetrate to the
basal layer of the epidermis, and mutations from oxidative
damage may significantly contribute to photocarcinogenesis
(16,17). UV radiation also contributes to carcinogenesis
through interactions with other cellular components, such as
cell signalling molecules (18) or immunoregulatory proteins,
by causing cell injury and inflammation (19–21).
Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon
and an environmental pollutant found in cigarette smoke, burnt
food and smoke from the burning of fossil fuels. BaP can be
absorbed by human skin (22) and metabolized, producing,
amongst other molecules, the ultimate carcinogen BaP diol
epoxide (BPDE) (23,24). BPDE binds to DNA, most commonly to guanines (25), forming bulky adducts that induce
GC!TA transversions (26). BaP has been implicated in the
development of skin cancer when combined with UV exposure.
Combined tobacco smoke and sunlight exposure is associated
with squamous cell carcinomas (27), and psoriasis patients
given coal tar therapy (which contains BaP) followed by
UVB radiation have an increased risk of skin cancer (28).
These observations suggest that combined exposure of
human cells to BaP and UV radiation may be much more
carcinogenic than would be expected from the effects of each
agent alone, and this hypothesis is supported by recent investigations. We have previously shown that binary exposure
of plasmid DNA to BPDE followed by UV radiation caused
a synergistic increase in single-strand breaks and mutation
frequency compared with the equivalent individual treatments
(29,30); and exposure of lung epithelial cells to BaP followed
by UV caused a more-than-additive increase in DNA damage
compared with each agent alone (31). However, the effects of
UV in combination with BaP have yet to be investigated in the
relevant cell type for skin carcinogenesis. Therefore, the aim of
this study was to elucidate the effects of UVA, UVB and UVC
radiation, alone and in combination with BaP exposure, on
normal human keratinocytes (NHKs) in vitro, by measuring
cell proliferation, DNA damage, and cell cycle perturbations.
To whom correspondence should be addressed. Tel: +44 113 3437763; Fax: +44 113 3436603; Email: [email protected]
# The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
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R.A.Crallan, E.Ingham and M.N.Routledge
Materials and methods
Cell culture
NHKs were isolated from paediatric foreskin samples, obtained with informed
parental consent and Local Ethics Committee approval, using a protocol adapted from Walters et al. (32). Foreskins were collected in Hank’s Balanced
Salt Solution (HBSS) containing 1% (v/v) penicillin/streptomycin (Invitrogen,
Paisley, UK) and stored at 4 C. Samples were washed three times in HBSS,
dissected into small pieces and incubated with 2 mg/ml dispase (Invitrogen) for
1 h at 37 C. The epidermis was peeled from the dermis, finely minced in 13
trypsin–EDTA (0.05% trypsin, 0.53 M EDTA, Invitrogen) and incubated
for 15 min at 37 C. Trypsin was deactivated with 10 mg/ml trypsin inhibitor
(TI, Sigma, Poole, UK) and cells harvested by centrifugation at 100 g for 5 min.
The isolated NHKs were resuspended in 5 ml defined keratinocyte serumfree medium (KSFM, Invitrogen), counted using a haemocytometer and
seeded onto 25 cm2 tissue culture flasks (Nunc, Fisher Scientific, Leicester)
coated with 0.67 mg/cm2 collagen (type IV, Sigma) at a density of at least 1 3
106 cells/flask. NHKs isolated in a similar manner and pooled from at least
three donors were also purchased from Invitrogen. NHKs were cultured in
KSFM at 37 C and 5% CO2 in air and the medium was changed every 2–3 days.
Sub-confluent cultures were passaged using 1 3 trypsin–EDTA, collected into
KSFM containing 2.5 mg/ml TI, harvested by centrifugation and re-seeded at
a 1:3 ratio in KSFM.
Cell based assays were repeated on at least three NHK cultures established
from independent donors.
Cell treatments
Sub-confluent NHK cultures were exposed to 1–50 mM BaP (Sigma), dissolved
in dimethyl sulfoxide (DMSO) and diluted in KSFM (final concentration of
DMSO <0.1%), for 24 h under normal culture conditions. For treatment of
cells with UV, culture medium was replaced with phosphate-buffered saline
(PBS) and culture dishes placed on an ice block. UVA exposure (365 nm) was
carried out with a UVL-26 lamp (UVP, Cambridge, UK) at a dose rate of
2 mW/cm2 for 50 s to 25 min; UVB (312 nm) and UVC (254 nm) irradiations
were carried out with a LF206MS UV lamp (UVP) at dose rates of 0.2 mW/cm2
for 5–50 s and 0.1 mW/cm2 for 2–10 s, respectively. For combined exposures,
cells were exposed to 5 mM BaP for 24 h, washed twice with PBS and then
exposed to 1 kJ/m2 UVA, 100 J/m2 UVB or 10 J/m2 UVC in PBS.
MTT cell viability assay
A quantitative determination of cell viability was based on the ability of
metabolically active cells to convert yellow formazan MTT salt to insoluble
blue crystals. NHK cells were seeded into collagen-coated 96-well plates
at 6 3 103 cells/cm2, which had been previously determined to allow NHK
cells to reach confluency after 7–10 days. Cell viability was assessed before
treatment (T0) and at various time-points after treatment, up to 7 days, by
adding 0.5 mg/ml MTT (Sigma) to each well and incubating at 37 C for 4 h.
The formazan crystals were dissolved in DMSO and the absorbance at 570 nm
was measured on a Multiskan Spectrum plate reader (Thermo Labsystems).
Cell cycle analysis by flow cytometry
The cell cycle profile of NHK cells was analysed by measuring the DNA
content of nuclei stained with propidium iodide (33), using DNA Staining
Reagent kit (Sigma). Actinomycin D (5 nM) was added to the cells as a positive
control for G1 arrest. Attached cells were lifted and combined with floating
cells 16 h after treatment and stained following the manufacturer’s protocol.
Briefly, 1 3 105 cells were washed twice with citrate buffer, digested with
trypsin and RNAse then stained with propidium iodide for 10 min at RT. The
DNA content of 10 000 cells/sample was analysed using a FACSCaliber flow
cytometer and CELLQuest software (Becton Dickinson, Oxford, UK).
Comet assay
For comet analysis of DNA damage, 1 3 106 cells/ml KSFM were collected
immediately after treatment and viability of the collected cells was assessed by
trypan blue exclusion, to ensure that viability amongst the assayed cells was
sufficiently high (at least 70%) for analysis in the comet assay. Cell suspension
(60 ml) was added to 300 ml 1% (w/v) low-melting-point agarose (Sigma) in
PBS kept at 37 C. Gel solution (162 ml) was pipetted onto duplicate microscope slides coated with 1% (w/v) normal-melting point agarose, flattened with
a coverslip and placed on ice for 30 s. Once the agarose had set, the coverslips
were removed and the slides were immersed in lysis solution [2.5 M NaCl,
100 mM EDTA, 10 mM Tris, pH 10, 1% (w/v) N-lauryl sarcosine, 1% (v/v)
Triton and 10% (v/v) DMSO] at 4 C for at least 1 h. Slides were transferred to
running buffer (0.3 M NaOH, 1 mM EDTA, pH 13) at 4 C for 40 min to allow
DNA to unwind, subjected to electrophoresis at 1 V/cm for 20 min, neutralized
in 0.4 M Tris, pH 7.5, for 5 min, air-dried and stained with 25 ml of 20 mg/ml
ethidium bromide (Sigma). Slides were stored on damp tissue paper at 4 C until
306
scoring. Comet formation was detected by fluorescent microscopy and quantified by computer assisted image analysis (Komet analysis software version 4,
Kinetic Imaging, Nottingham, UK). Fifty nuclei/slide were randomly selected
and percentage tail DNA measured. To detect oxidized purines, nuclei were
incubated with Fpg enzyme (a kind gift from Andrew Collins) after lysis: slides
were washed 3 3 5 min in enzyme buffer (40 mM HEPES, 0.1 M KCl, 0.5 mM
EDTA, 0.2 mg/ml BSA), whereafter 50 ml Fpg (1:3000; final concentration
0.16 mg/ml) or 50 ml enzyme buffer was added to each slide with a coverslip
and incubated at 37 C for 30 min prior to unwinding. The assay was completed
and analysed as described above.
32
P-postlabelling of DNA adducts
Genomic DNA was extracted from NHK cells after 24 h incubation with 5 mM
BaP (Qiagen blood and cell culture DNA mini kit, Qiagen, Crawley, UK). DNA
(5 mg) was analysed by 32P-postlabelling, as previously described (29). Radioactivity was visualized using a FLA-5000 phosphorimager (Fujifilm).
Statistical analysis
Results are expressed as means of three replicates 695% confidence intervals
(CIs), unless stated otherwise. Percentage data were first transformed (arcsin
transformation) before analysis by ANOVA using Instat (version 3.0,
Graphpad, San Diego).
Results
Cell viability
Cell survival was measured for 7 days after exposure to BaP
and UV, alone and in combination, using the MTT viability
assay (Figure 1). Exposure to 5 mM BaP for 24 h caused a
decrease in cell viability for the duration of the exposure,
followed by recovery to control levels. UV exposure alone
slightly reduced the growth rate of NHKs over the 7 day period,
with cultures reaching 75% of the control population viability.
After combined exposure to 5 mM BaP and 100 J/m2 UVB or
10 J/m2 UVC, no further effect on viability was observed.
However, combined exposure of NHKs to 5 mM BaP followed
by 1 kJ/m2 UVA caused a dramatic reduction in viability to
60% of the initial cell density and <10% of the final control
cell viability, with no recovery within 7 days. These cells
retained normal keratinocyte morphology but were resistant
to trypsin.
Cell cycle
The cell cycle profile was measured by labelling NHK cell
nuclei with propidium iodide and measuring DNA content by
flow cytometry. Untreated NHK cultures had a proliferating
cell cycle profile with 50% of cells in G1, 20% in S phase and
30% in G2 (Figure 2). Exposure to BaP or UVA had no effect
on cell cycle, but 100 J/m2 UVB and 10 J/m2 UVC caused
a significant accumulation of nuclei in G2 to 47.7% (61.8%,
P 5 0.0017) and 42.6% (62.2%, P 5 0.0128), respectively.
Pre-exposure to 5 mM BaP did not affect the UVC-induced G2
delay (39.6 6 3.3%, P 5 0.0392), but the UVB-induced delay
was abolished (34.1 6 1.0%, not significant compared with
control).
DNA damage in NHKs
Using the nuclease P1 enhanced 32P-postlabelling assay, we
demonstrated formation of DNA adducts in NHKs after 24 h
exposure to 10 mM BaP (Figure 3). The pattern of radioactivity
from NHK genomic DNA was similar to the pattern obtained
from plasmid labelled with BPDE as a positive control. The
‘hotspot’ on each chromatogram probably represents the major
adduct BPDE-N2-deoxyguanine. No adducts were detected in
DNA extracted from unexposed NHKs.
DNA damage (single and double strand breaks, alkali-labile
sites) in NHKs was measured by the comet assay. Doses that
caused little or no detectable DNA damage were chosen from
Combined exposure of keratinocytes to BaP and UV
Fig. 1. Cell viability over 7 days measured by MTT assay. Cells were seeded at low density and viability measured before treatment (0) and on days 1, 2, 4 and 7
after addition of BaP. BaP was added to cells in six replicate wells for 24 h. After removal, cells were washed twice with PBS before exposure to UV in PBS. Data
expressed as mean 6 SD (n 5 6) from one cell line, representative of three independent cell lines. Solid black line with diamonds, untreated NHKs; solid black line
with crosses, 5 mM BaP; dotted line with closed triangles, 1 kJ/m2 UVA; dotted line with open triangles, 5 mM BaP 11 kJ/m2 UVA; dashed line with closed
ellipses, 100 J/m2 UVB; dashed line with open ellipses, 5 mM BaP 1 100 J/m2 UVB; solid gray line with closed rectangles, 10 J/m2 UVC; solid gray line
with open rectangles, 5 mM BaP 1 10 J/m2 UVC.
Fig. 2. Cell cycle profiles generated by propidium iodide labelling of nuclei, with increasing DNA content along the x-axis versus number of nuclei on
the y-axis. (A) untreated NHKs; (B) 1 kJ/m2 UVA; (C) 100 J/m2 UVB; (D) 10 J/m2 UVC; (E) 5 mM BaP; (F) 5 mM BaP 1 1 kJ/m2 UVA;
(G) 5 mM BaP 1 100 J/m2 UVB; (H) 5 mM BaP 1 10 J/m2 UVC.
307
R.A.Crallan, E.Ingham and M.N.Routledge
A
B
C
Fig. 3. 32P-postlabelling of DNA. Chromatogram A shows plasmid DNA reacted with BPDE in vitro as a positive control. Chromatogram B shows the
pattern of adducts detected in genomic DNA extracted from NHKs immediately after exposure to 10 mM BaP for 24 h. Chromatogram C shows
NHK genomic DNA without BaP exposure as a negative control.
15
*
40
35
% tail DNA
30
†
25
‡
20
15
10
0.01% DMSO
5 mM BaP
*
0.01% DMSO
5 mM BaP
increase in % tail DNA
45
†
10
5
5
0
0
control
1kJ/m2 UVA
100 J/m2 UVB
10J/m2 UVC
Unirradiated
1 kJ/m2 UVA 100 J/m2 UVB
10 J/m2 UVC
Fig. 4. Mean percentage tail DNA in NHKs analysed by the comet assay
illustrating the effects of UV with and without BaP exposure on DNA
damage. Data expressed as mean 6 95% CI of three cell lines. P 5 0.0015
compared with 1 kJ/m2 UVA alone, †P 5 0.0379 and ‡P 5 0.0449 compared
with unirradiated control, by ANOVA.
Fig. 5. Increase in percentage tail DNA after Fpg incubation. Exposure to
5 mM BaP followed by 1 kJ/m2 UVA or 100 J/m2 UVB caused significantly
increased tail formation compared with each agent alone. Data expressed as
mean 6 95% CI of three cell lines. P 5 0.0034, †P 5 0.0011 compared with
UV radiation alone, by ANOVA.
preliminary dose–response studies. Exposure to 5 mM BaP for
24 h or 1 kJ/m2 UVA did not increase tail formation above
background levels (<10% tail DNA), whereas 100 J/m2 UVB
and 10 J/m2 UVC induced a small, but significant, increase in
percentage tail DNA to 17.9 6 5.1% and 13.8 6 2.6% respectively (Figure 4). Exposure to 5 mM BaP followed by 1 kJ/m2
UVA radiation caused a very significant increase in percentage
tail DNA to 35.1% (62.5%, P 5 0.0018); this effect was not
observed if the treatment order was reversed (data not shown).
BaP pre-exposure also increased tail formation after UVB
radiation but to a much lesser extent than UVA, and BaP preexposure had no effect on tail formation by UVC radiation.
Although combined exposure to BaP followed by UVA caused
a dramatic reduction in viability, the majority of dead cells
were resistant to trypsin and were not harvested for comet
analysis. Trypan blue exclusion of harvested cells showed
that 76.6% (613.6%) were viable after combined treatment.
BaP followed by 1 kJ/m2 UVA, there was a significant increase
of 11.2 6 2.9% tail DNA compared with UVA exposure alone
(P 5 0.0034). Combined exposure to 5 mM BaP followed by
100 J/m2 UVB also caused a significant increase of 9.3 6 2.2%
tail DNA (P 5 0.0011). Pre-exposure to BaP did not significantly increase the levels of oxidative lesions detected after
UVC radiation.
Oxidative DNA damage
Incubation of comet slides with Fpg enzyme enabled the detection of oxidized purines, by conversion of the base damage into
single strand breaks. The increase in percentage tail DNA after
Fpg incubation compared with slides incubated with enzyme
buffer alone is shown in Figure 5. There was a small increase in
percentage tail DNA in control cells owing to basal levels of
oxidative damage. After combined exposure of NHKs to 5 mM
308
Discussion
Human epithelial cells are exposed in vivo to a myriad of
genotoxic agents, but limited consideration has been given to
the effect of combined exposures to these agents. Here we have
investigated the combined effects of two common mutagens,
BaP and UV light, on NHKs in vitro and showed that the
effects of combined exposure differ, depending on the wavelength of the UV source. The most dramatic effects were
observed with UVA, the principal component of sunlight.
NHKs in vitro were highly sensitive to UVA radiation after
BaP exposure, and the marked reduction in viability was
accompanied by increased resistance to trypsin, an effect
which has previously been described in breast carcinoma
cells (34). Enhanced cytotoxicity after coexposure to BaP and
UVA has been reported in human skin fibroblasts (35) and
CHO cells (36). Binary exposure to BaP followed by UVA
also caused significantly enhanced DNA damage, both strand
Combined exposure of keratinocytes to BaP and UV
breaks and oxidative lesions, even though each agent alone did
not induce any detectable effects. Increased ROS production
and oxidative lesions after combined BaP/UVA treatment have
been described in several human cell types in vitro (35,37,38),
in mouse skin in vivo (39) and in calf thymus DNA (40).
However, many of these studies looked at simultaneous, rather
than sequential, BaP/UVA exposure. Our results suggest that
the presence of BaP or BPDE-adducts synergistically enhances
the induction of DNA damage by UVA light, leading to cell
death, and may play a role in development of mutations in
human skin cells. Indeed, combined exposure of mouse embryonic fibroblasts to non-mutagenic doses of BaP and UVA was
recently demonstrated to enhance mutation frequency (41).
The mechanism of this enhancement, however, is still
unclear. BPDE–DNA adducts formed during the 24 h incubation with BaP may be photosensitive, leading to DNA strand
breakage on exposure to UVA light, which has been shown
to occur in oligonucleotides containing a BPDE-N2-guanine
adduct (42). Alternatively, as BaP absorption lies within the
UVA and visible light spectrum and BaP has been shown to
be photomutagenic (43), BaP, or metabolites of it, remaining
inside the keratinocytes after washing may become activated
by UVA light and subsequently cause damage.
In contrast to UVA, UVB had no effect on cell viability and
did not increase strand breakage in human keratinocytes
after BaP pre-exposure. This contradicts a previous study that
reported enhanced DNA damage by the comet assay in bovine
retinal pigment epithelial cells after combined exposure to BaP
and UVB (44). BaP/UVB treatment did, however, induce
increased oxidative DNA damage compared with BaP or UVB
exposure alone. This observation could be important in understanding the increased incidence of skin cancer in psoriasis
patients who received coal tar therapy followed by UVB
irradiation as treatment for their disorder.
Both UVB and UVC induced G2 arrest in NHKs, as previously described (45,46). Interestingly, the UVB-induced G2
arrest of keratinocytes was abrogated by BaP pre-exposure.
BaP has been reported to have ‘stealth’ properties and can
avoid inducing cell cycle arrest (47,48): in this instance, it
appeared that BaP inhibited the recognition of UVB-induced
DNA damage, which could have significant long-term effects
on the genetic stability of the cells.
The response of NHKs to UVC was not affected by BaP preexposure. This is surprising as we have previously shown that
plasmid exposed to BPDE followed by UVC has a greater
increase in single strand breaks and mutation frequency than
plasmid treated with BPDE and UVB (29), and lung epithelial
cells treated with BaP followed by UVC have enhanced DNA
damage (31). These differences may be due to cell-specific
properties, which is why it is important to investigate the
effects of genotoxin exposure in the relevant cell type.
The effects of combined exposure of NHKs to BaP and UV
light are wavelength-dependent. BaP and UVA induced considerable cell death and DNA damage. This finding could be of
particular significance due to the prevalence of UVA in sunlight and the penetration of UVA to the basal layers of the skin,
especially as the dose used in this investigation is equivalent to
3 min of sun exposure on a sunny summer’s day in northern
Europe (49). BaP and UVB exposure increased the induction of
oxidative damage and bypassed UVB-induced G2 cell cycle
arrest. UVB is also present in sunlight, and the dose used in this
study equates to just over 1 min of sunlight exposure. The
mechanisms of these responses are unclear but the importance
of studying the effects of agents in combination is highlighted.
Our results also have implications for the relative contributions
of UVA and UVB to skin cancer development. UVB is more
efficient at causing CPDs than UVA; for example, Woollons
et al. (13) showed that in cultured keratinocytes the majority
(75%) of CPDs are induced by the 0.8% UVB content in a
UVA sunlamp. However, the upper layers of human skin in
vivo will filter out more UVB than UVA and may effect these
relative contributions. The data presented here suggest that
UVA may have a much more prominent role in skin carcinogenesis because it interacts with other agents, like BaP, in the
environment. Thus, ‘safe’ sun exposure recommendations may
need to be reduced, especially in polluted areas, in order to
prevent skin cancer development. Also, as BaP exposure
caused oxidative damage after both UVA and UVB radiation,
the inclusion of anti-oxidants in sun cream, which has been
suggested to have anti-ageing benefits, may further contribute
to skin cancer prevention.
Acknowledgements
We thank Andrew Collins for the gift of Fpg enzyme. This work was funded by
Yorkshire Cancer Research; and supported by a Yorkshire Cancer Research
grant L299 awarded to M.N.R. and E.I.
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Received on March 23, 2005; revised on May 19, 2005;
accepted on May 20, 2005