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Guanine sulphinate is a major stable product of
photochemical oxidation of DNA 6-thioguanine
by UVA irradiation
Xiaolin Ren
1,2
, Feng Li
1
, Graham Jeffs
2
, Xiaohong Zhang
1,2
, Yao-Zhong Xu
2
and
Peter Karran
1,
*
1
Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD and
2
Department of Chemistry, the Open University, Walton Hall, Milton Keynes MK7 6AA, UK
Received October 28, 2009; Revised November 18, 2009; Accepted November 24, 2009
ABSTRACT
The DNA of patients taking the immunosuppres-
sant and anticancer drugs azathioprine or
6-mercaptopurine contains 6-thioguanine (6-TG).
The skin of these patients is selectively sensitive
to ultraviolet A radiation (UVA) and they suffer
an extremely high incidence of sunlight-induced
skin cancer with long-term treatment. DNA 6-TG
interacts with UVA to generate reactive oxygen
species, which oxidize 6-TG to guanine sulphonate
(G
SO3
). We suggested that G
SO3
is formed via the
reactive electrophilic intermediates, guanine
sulphenate (G
SO
) and guanine sulphinate (G
SO2
).
Here, G
SO2
is identified as a significant and
stable UVA photoproduct of free 6-TG, its
20-deoxyribonucleoside, and DNA 6-TG. Mild
chemical oxidation converts 6-TG into G
SO2
, which
can be further oxidized to G
SO3
—a stable product
that resists further reaction. In contrast, G
SO2
is con-
verted back to 6-TG under mild conditions. This
suggests that cellular antioxidant defences might
counteract the UVA-mediated photooxidation of
DNA 6-TG at this intermediate step and ameliorate
its biological effects. In agreement with this possi-
bility, the antioxidant ascorbate protected DNA 6-TG
against UVA oxidation and prevented the formation
of G
SO3
.
INTRODUCTION
Reactive oxygen species (ROS) are hazardous to cells.
They cause damage to DNA, proteins and membranes.
Cells are well adapted to the levels of ROS produced
in the normal course of aerobic metabolism and are
equipped with powerful antioxidant defences and repair
systems to counteract the effects of inevitable damage
to DNA. Exogenous sources of ROS can perturb this
homeostasis and excess ROS create a condition of oxi-
dative stress in which cellular defences are overwhelmed
and cellular constituents are damaged. Potentially
damaging ROS are produced when cells are exposed to
ultraviolet A (UVA, wavelengths 320–400 nm) radiation.
This reaction, which occurs at high (>100 kJ/m
2
) UVA
doses, involves the absorption of UVA energy by
cellular chromophores (1). DNA, a major target for
damage by ROS, is a UVC and UVB chromophore but
does not absorb the longer wavelength UVA to a signifi-
cant degree. UVA comprises >90% of incident ultraviolet
radiation at the earth’s surface and the inability of DNA
to absorb UVA, and thereby generate ROS, is an impor-
tant factor in minimizing sunlight-induced damage to
DNA (2).
The thiopurines azathioprine (Aza), 6-mercaptopurine
(6-MP), and 6-thioguanine (6-TG) are immunosup-
pressant, anti-inflammatory, and anticancer drugs (3).
They are all metabolized to 6-TG nucleotides (TGNs)
and cause the incorporation of 6-TG into DNA (4).
Unlike the canonical DNA bases, 6-TG is a strong UVA
chromophore. When 6-TG is irradiated with UVA
(340 nm) reactive oxygen species (ROS) and, in particular,
singlet oxygen (
1
O
2
) are generated (5,6). DNA 6-TG and
the TGN pool are both significant sources of ROS when
thiopurine-treated cells are UVA irradiated (7). The for-
mation ROS from 6-TG within DNA itself is potentially
extremely hazardous and
1
O
2
is an acknowledged source
of oxidized DNA bases, DNA strand breaks (8) and oxi-
dation of DNA associated proteins (9). In addition to this
danger to normal cellular constituents, the low oxidation
potential of 6-TG makes it a preferred target for oxidation
and this compounds the hazard of photochemically
generated ROS. We have previously shown that this
*To whom correspondence should be addressed. Tel: þ44 (0)170 762 5870; Fax: þ44 (0)170 762 5801; Email: peter.karran@cancer.org.uk
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
1832–1840 Nucleic Acids Research, 2010, Vol. 38, No. 6 Published online 21 December 2009
doi:10.1093/nar/gkp1165
ßThe Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
favoured oxidation of DNA 6-TG generates guanine
sulphonate (G
SO3
) a highly effective block to replication
and transcription (5,6,10).
Long-term therapy with thiopurines, for example, in
immunosuppressed organ transplant recipients treated
with Aza, is associated with a very high incidence of
skin cancer for which sunlight exposure is a contributory
factor (11). The skin of patients undergoing Aza treatment
contains DNA 6-TG and, as a consequence, is selectively
sensitive to the induction of erythema by UVA (12). This
UVA-selective photosensitivity suggests that photochem-
ically induced DNA damage is physiologically relevant.
In view of the huge incidence of skin cancer in this
patient group, we have examined in more detail the
photochemistry of 6-TG and DNA 6-TG in order to
define the DNA lesions likely to be present in the skin
of Aza-treated patients exposed to sunlight. The UVA
doses we use in these in vitro studies fall well within the
range to which patients might be exposed on an averagely
sunny summer day in Northern Europe (13). DNA
guanine sulphinate (G
SO2
) as a quantitatively major
product of the photochemical oxidation of DNA 6-TG
and an intermediate in the formation of the previously
described DNA G
SO3
(5). We demonstrate that G
SO2
is converted back to 6-TG under relatively mild condi-
tions, suggesting that cellular reducing agents may also
provide some protection against sunlight by reversing
photochemical damage to DNA 6-TG. In view of the
involvement of ROS in the formation of these DNA
lesions, we also examined the protective potential of the
dietary vitamin ascorbate (vitamin C)—an acknowledged
antioxidant in skin (14). We report that ascorbate prevents
DNA damage and protects 6-TG, and DNA 6-TG, from
UVA-induced oxidation.
EXPERIMENTAL PROCEDURES
Chemicals
6-thioguanine (6-TG), deoxyguanosine (dG), ascorbate
(vitamin C), sodium sulphide (Na
2
S:9H
2
O), hydrogen
peroxide 30%, acetic acid, magnesium monoperox-
yphthalate (MMPP), Fe(NH
4
)
2
(SO
4
)
2
, Rose Bengal and
iodine (I
2
) were obtained from Sigma Aldrich (Dorset,
UK). Oligodeoxyribonucleotides were from Oligos Etc.,
Inc. (Wilsonville, USA). G
SO3
was prepared as described
(5). G
SO2
was prepared according to a published protocol
(15). Guanine-6-thioguanine (G
S
G) was prepared as
before (6).
Chromatography
Four different systems of reverse-phase analyses for
modified 6-TG and 6-TGdR were used.
System 1. Bases were separated by high-performance
liquid chromatography (HPLC) on a Waters dC
18
(Atlantis 3 mm, 150 2.1 mm) column using a Waters
2695 Alliance system equipped with photodiode array
and Waters 474 dual monochromator fluorescence
detector. Column eluates were monitored simultaneously
by absorbance and fluorescence. Elution was with
three solvents: A ¼methanol; B ¼water; C ¼100 mM
KH
2
PO
4
, pH 6.7. Flow rate was increased from 0.2 to
0.25 ml/min during the first 23 min, and at 0.25 ml/min
thereafter. Solvent C was kept constant at 5% for
23 min and 0% thereafter. A gradient 0–20% Solvent A
was applied during the first 10 min. Solvent A was
increased to 80% over the next 10 min and to 90% over
the next 3 min and maintained at 90% for 7 min.
System 2. Bases were separated by HPLC on a Waters
C
18
column (Xterra MS 3.5 mm, 150 2.1 mm) equipped
and monitored as System 1. Solvents A and B were as
System 1, Solvent C ¼10 mM KH
2
PO
4
, pH 6.5. Flow
rate was constant at 0.2 ml/min. Solvent C was kept at
20%. A gradient 0–20% Solvent A was applied during
the first 10 min. Solvent A was increased to 80% over
the next 10 min and maintained at 80% for 5 min.
System 3. Column, solvents A, B and C and eluates
monitored were as System 1. Flow rate was 0.2 ml/min.
Solvent C was kept constant at 5%. Solvent A was
increased from 0% to 20% during the first 10 min, then
increased to 80% during the next 10 min and maintained
at 80% for 5 min.
System 4. 2-deoxyribonucleosides were separated by
HPLC on a Waters Symmetry C
18
Reversed Phase
column as described (5). 6-TGdR and dG
SO2
were
quantified by A
342
,dGbyA
260
and dG
SO3
by its fluores-
cence (Ex ¼320 nm; Em ¼410 nm). Elution was with two
solvents: A ¼10 mM KH
2
PO
4
pH 6.7; B ¼methanol.
Flow rate was constant at 0.5 ml/min. A gradient
100–90% Solvent A was applied during the first 10 min,
then 90–60% during the next 10 min, maintained at 60%
for 2.5 min. This was followed by a sharp gradient
60–100% Solvent A for 0.5 min and constant at 100%
for 17 min.
Nuclear magnetic resonance
Nuclear magnetic resonance (NMR) experiments
were conducted at 300 MHz from JOEL (JNM-LA 300,
FT NMR) and 400 MHz from JOEL (JNM-EX 400,
FT NMR). Samples were dissolved in DMSO-d6 and
chemical shifts were adjusted with reference to an
internal standard tetramethylsilane (TMS).
Mass spectroscopy
Mass spectroscopy (MS) analysis was performed on
a Fisons Instruments (VG BioTech, Altrincham, UK)
Quattro Electrospray ionization mass spectrometer.
Samples were dissolved in 50:50 MeOH:Water (ESI
negative mode). High-resolution mass spectra (ESI
negative mode) were obtained by the EPSRC Mass
Spectrometry Service Centre, University of Swansea.
Fluorescence spectrometry
Fluorescence emission and excitation spectra were
obtained using a FLUOROMAX-P spectrofluorometer,
Jobin Yvon Horiba Inc. UK.
Nucleic Acids Research, 2010, Vol. 38, No. 6 1833
UVA irradiation
6-TG, 6-TGdR, oligonucleotides and genomic DNA were
irradiated at a dose rate of 0.07 kJ/m
2
/s (for 6-TG) or
0.1 kJ/m
2
/s (for all other samples) using a UVH 254
lamp (UV Light Technology, wavelength range 320–400).
Digestion of DNA to 20-deoxyribonucleosides
Genomic DNA was extracted using the Wizard Genomic
DNA purification kit (Promega, Madison, WI, USA)
from cells grown in medium containing 1 mM 6-TG for
24 h. Standard digestion to nucleosides for single-stranded
oligonucleotides and genomic DNA was with nuclease P1
at pH 4.7 followed by shrimp alkaline phosphatase as
described (10).
RESULTS
6-TG photoproducts
UVA irradiation of 6-TG in 0.1 mM aqueous solution
causes a rapid loss of its characteristic A
342
(5). HPLC
separation of the 6-TG photoproducts after 21 kJ/m
2
revealed four novel peaks with absorbance at 320 nm
eluting at 2.8, 3.5, 6.5 and 14.8 min (Figure 1A, ii). The
small amount of unchanged 6-TG eluted at 9 min.
The compounds eluting at 2.8 and 14.8 min were the
previously identified as the highly fluorescent
(l
ex
¼324 nm, l
em
¼408 nm) G
SO3
and the 6-TG dimer,
G-S-G, respectively. Their identities were confirmed by
co-elution with the authentic compounds. A small peak
at 6.5 min was identified as guanine by its UV spectrum.
The formation of these photoproducts was examined
over a range of UVA doses. HPLC analysis indicated
that the photoproduct eluting at 3.5 min was generated
rapidly and that its formation preceded that of detectable
amounts of G
SO3
and G-S-G (Figure 1B). Comparison of
its absorbance spectrum with published data (15) sug-
gested that this initial product was guanine-6-sulphinate,
G
SO2
. Authentic G
SO2
was prepared by I
2
-mediated
oxidation of 6-TG according to a published protocol
(15) and crystallized. The product was not fluorescent,
as expected, and its absorbance spectrum together
with mass and NMR spectrometry confirmed that it was
G
SO2
(Supplementary Figure S1). Authentic G
SO2
co-eluted from HPLC exactly coincident with the
3.5-min 6-TG photoproduct (Figure 1A, iii and iv).
Together, the four identifiable UVA photoproducts:
G
SO2
,G
SO3
, G, and G-S-G accounted for >90% of the
6-TG that was destroyed by UVA. G
SO2
was the most
abundant initial product. These findings indicate that
G
SO2
is a relatively stable intermediate in UVA-mediated
6-TG oxidation. It can be further oxidized to G
SO3
.
Guanine and the G-S-G dimer are also generated as
minor photoproducts.
Sequential oxidation of 6-TG was investigated further
using MMPP which oxidizes 6-TG to G
SO3
(6). Figure 1C
shows that when 6-TG was treated for 10 min at room
temperature (RT) with equimolar MMPP, some 6-TG
remained unchanged and G
SO2
was the only product
detectable by A
320
. When MMPP was in 2-fold molar
excess over 6-TG, approximately equal amounts of G
SO2
and G
SO3
were produced. Under stronger oxidising con-
ditions (6-TG:MMPP ¼1:3), 6-TG was stoichiometrically
converted to G
SO3
. Purified G
SO2
was quite stable and
remained unchanged by overnight incubation at 20in
aqueous solution (data not shown). It could be,
however, oxidized by MMPP. A 10-min treatment at RT
with a limiting MMPP concentration (G
SO2
:MMPP ¼
1: 0.5) converted about 80% to G
SO3
. No other oxidation
products were detected and higher MMPP concentrations
(G
SO2
:MMPP ¼1: 1), induced a stoichiometric conversion
to G
SO3
(Figure 1D).
We conclude that G
SO2
—which has absorbance
at 320 nm but is not fluorescent (Supplementary
Figure S1)—is the first stable product of 6-TG photo-
oxidation. G
SO2
,G
SO3
, guanine and G-S-G together
account quantitatively for all the 6-TG destroyed by
UVA. G
SO2
is produced by chemical oxidation under rel-
atively mild conditions and, although stable, it easily
undergoes further oxidation to G
SO3
.
The stoichiometry of oxidation of the 6-TG
20-deoxyribonucleoside (6-TGdR) was similar and dG
SO2
was the major UVA photoproduct when an aqueous
solution of 6-TGdR (20 mM) was irradiated. Approxi-
mately 50% was destroyed by 15 kJ/m
2
and 90% by
45 kJ/m
2
. Figure 2 shows that at 15 kJ/m
2
,dG
SO2
accounted for around 90% of the destroyed 6-TGdR. At
higher doses, both dG
SO3
and dG were formed. These
minor products were present in similar amounts and
each accounted for 10–20% of the destroyed 6-TGdR at
45 kJ/m
2
UVA. There was no indication of the forma-
tion of other significant photoproducts at least up to
135 kJ/m
2
, the highest dose used (data not shown).
dG
SO3
,dG
SO2
and dG together accounted for 90% of
the 6-TGdR destroyed by UVA. The minor fraction of
input 6-TGdR that was unaccounted for was most likely
converted to the nucleoside dimer analogous to G-S-G as
we also observed a late-eluting product with A
342
which
might be consistent with this compound (Supplementary
Figure S2). A final confirmation and quantification of this
structure awaits synthesis of the authentic nucleoside
dimer.
To approach the identification of DNA 6-TG photo-
products, we first examined the effect of UVA on single-
stranded oligonucleotides containing a single 6-TG. Since
our previous findings indicated that dG was a potential
photoproduct, we first examined the effect of UVA on an
8-mer comprising 7As and a single 6-TG (A
7
TG
1
). This
permitted the accurate quantitation of any dG formed.
Irradiated single-stranded oligos were digested with
nuclease P1 followed by alkaline phosphatase and the
20-deoxyribonucleosides were quantified as above. In an
attempt to minimize losses due to the known acid
lability of the photoproducts (15), nuclease digestion of
irradiated 8-mers was first carried out at pH 7. Under
these conditions, photoproduct recovery was poor and
over a range of UVA doses (5–45 kJ/m
2
) only around
30% of destroyed 6-TG could be accounted for by
dG
SO3
,dG
SO2
and dG. Previously unidentified photo-
products were generated in UVA-dependent fashion
(Supplementary Figure S3). This appeared to be the
1834 Nucleic Acids Research, 2010, Vol. 38, No. 6
product of incomplete P1 nuclease digestion under
these sub-optimal conditions. Digestion was also incom-
plete when the irradiated oligo was incubated under
optimal conditions (6 h at pH 8.0) for benzonase
(Sigma), phosphodiesterase I and alkaline phosphatase
(data not shown). These findings suggest that some
6-TG photoproducts are somewhat resistant to enzymatic
digestion.
Under conditions that were optimal for nuclease P1,
pH 4.7 and 50C, digestion of irradiated (A
7
TG
1
) was
Figure 1. 6-TG/UVA photoproducts. (A) Identification of G
SO2
as a major photoproduct. 6-TG (0.1 mM in aqueous solution) was irradiated with
21 kJ/m
2
UVA. Products were separated by HPLC System 1 as described in ‘Experimental Procedures’ section and the eluate monitored at 320 nm.
(Top panel) Unirradiated 6-TG. (Second panel) 6-TG after 21 kJ/m
2
UVA. (Third panel) As second panel, with authentic G
SO2
added before HPLC.
(Bottom panel) Authentic G
SO2
(prepared by mild I
2
oxidation of 6-TG) alone. (B) Quantitation of photoproducts. 6-TG [as in (A)] was irradiated
with UVA at a dose rate of 0.07 kJ/m
2
/s. Samples were removed and analysed on HPLC. The four peaks of 320 mn absorbance with retention times
of 2.8 min (filled square), 3.5 min (open circle), 6.5 min (filled triangle) and 14.8 min (open triangle) were identified as G
SO3
,G
SO2
, G and G-S-G as
described in the text. Unaltered 6-TG (filled circle) eluted at 9 min. Quantitation was at the absorbance maximum for each product and by
comparison to authentic standard compounds. G
SO3
(A
325
), G
SO2
(A
320
), G (A
273
), 6-TG (A
340
) and G-S-G (A
331
). Products are expressed mole
% of unirradiated 6-TG. A representative of three independent experiments is shown. (C) 6-TG oxidation by MMPP. 6-TG (0.1 mM) was treated for
10 min at 20C with MMPP at the final molar ratio indicated. Products were separated by HPLC System 2 and eluates monitored at 320 nm.
Unchanged 6-TG elutes at 9 min, G
SO2
at 3.5 min and G
SO3
at 3.0 min. (D)G
SO2
oxidation by MMPP. G
SO2
(0.1 mM) was treated for 10 min at 20
with MMPP at the molar ratios shown. Products were separated by HPLC System 3.
Nucleic Acids Research, 2010, Vol. 38, No. 6 1835
complete and all of the destroyed 6-TG was accounted for
by dG
SO3
,dG
SO2
and dG. Under these conditions, dG
appeared to be the major product (Figure 3A). In view
of the known lability of G
SO2
under acid conditions, it
seemed likely that some of the dG had been generated
from dG
SO2
during DNA digestion at pH 4.7. To investi-
gate this, UVA-irradiated 6-TGdR was subjected to the
same conditions of temperature and pH. Figure 3B shows
that this treatment resulted in loss of a significant frac-
tion of dG
SO2
. The major part of the missing dG
SO2
was
recovered as dG with a small amount converted to
6-TGdR. dG
SO2
formation in UVA irradiated oligos was
corrected for this artefactual destruction of dG
SO2
(see
legend to Figure 3C). When this correction for artefactual
conversion of dG
SO2
to dG was applied, dG
SO2
and dG
SO3
together accounted for 95% of the destroyed 6-TG.
dG
SO2
was the major photoproduct (75–90% of the
total) (Figure 3C).
dG
SO2
was also the major photoproduct in double-
stranded DNA (Table 1). DNA uniformly substituted
with 6-TG to 2% of G, was purified from cells that
had been grown for 24 h in the presence of 6-TG. After
UVA irradiation, heat denaturation, digestion with
nuclease P1 under standard conditions (pH 4.7, 1 h, 50)
and treatment with alkaline phosphatase, dG
SO3
appeared
to be the predominant photoproduct detected by HPLC
(it was not possible to quantify any increase in dG). The
two oxidized forms, dG
SO3
and dG
SO2
, together
accounted for 20% of the starting DNA 6-TG and no
other significant peaks of absorbance at 260, 320 or
342 nm were observed. When corrected for losses of
labile dG
SO2
under the conditions of enzyme digest,
however, around 90% of input DNA 6-TGdR was
accounted for and dG
SO2
was again the major
photoproduct. These data indicate that dG
SO3
and
dG
SO2
comprise almost all of the UVA photoproducts
of DNA 6-TGdR. At the relatively low doses of UVA
that we use (5–15 kJ/m
2
), dG
SO2
accounts for essentially
all of the destroyed 6-TGdR and that dG
SO3
and dG are
formed to a minor extent. At higher doses, a measurable
fraction (10–20% of the total) is oxidized to dG
SO3
and a
similar fraction of dG is formed. These photochemical
reactions are largely independent of the 6-TG context
and the proportions of photoproducts are similar for the
base, the 20-deoxyribonucleoside and for 6-TG in single-
or double-stranded DNA.
Figure 3. UVA oxidation of 6-TG in oligonucleotide. (A) Single-
stranded 8-mer oligo A
7
TG
1
(AAAAXAAA where X¼6-TG; 50-mM
aqueous solution) was irradiated with UVA, then digested to
20-deoxyribonucleosides by nuclease P1 (1 h, 50, pH 4.7) followed by
alkaline phosphatase as described in ‘Experimental Procedures’ section.
Products were separated and quantitated as described in legend
to Figure 2. Products are expressed mole % of unirradiated 6-TG.
A representative of three independent experiments is shown.
(B) 6-TGdR (20 mM aqueous solution) was irradiated with 15 kJ/m
2
UVA, half the sample was immediately analysed by HPLC. The other
half was subjected to the same conditions of temperature and pH as for
nuclease P1 (pH 4.7, 1 h at 50C) and alkaline phosphatase digestion
before HPLC analysis. The means and SD of three independent exper-
iments are shown. (C) Corrected photoproduct yield. The amount of
dG
SO2
destroyed under acidic conditions was calculated based on yield
of dG from the irradiated A
7
TG
1
and the known dG formation after
UVA irradiation of 6-TGdR at pH 7. The total yield of dG
SO2
¼dG
SO2
measured þdG
SO2
calculated from (dG measured in A
7
TG
1
oligo
digested at pH 4.7—dG measured from irradiated 6-TGdR at neutral
pH from Figure 2).
Figure 2. UVA oxidation of 6-TG 20-deoxyribonucleoside. 6-TGdR
(20 mM aqueous solution) was irradiated with UVA in neutral condi-
tions (10 mM Tris–HCl pH 7.5). Products were immediately separated
by HPLC System 4. Quantitation of unaltered 6-TGdR (filled circle)
and dG
SO2
(open circle) by their A
342
,dG
SO3
(filled square) by its
fluorescence at 410 nm, dG (filled triangle) by its A
260
were by compar-
ison to authentic standard compounds. Products are expressed mole %
of unirradiated 6-TG. A representative of three independent experi-
ments is shown.
1836 Nucleic Acids Research, 2010, Vol. 38, No. 6
Mechanism of 6-TG photooxidation
6-TG is a Type II UVA photosensitizer (6). We therefore
investigated whether
1
O
2
generated photochemically by
Rose Bengal and visible light mediated the stepwise con-
version of 6-TG to G
SO3
via G
SO2
. When a mixture of
6-TG (0.1 mM) with Rose Bengal (0.5 mM) was irradiated
with 360-kJ visible light, 90% of the 6-TG was oxidized.
Both G
SO2
and G
SO3
were produced (Figure 4A).
Rose Bengal plus visible light treatment of authentic
G
SO2
provided further evidence for stepwise oxidation
from 6-TG to G
SO3
. When combined with a 5-fold
molar excess of Rose Bengal and irradiated with differ-
ent doses of visible light, G
SO2
was oxidized to G
SO3
(Figure 4B, i-iii). In contrast, G
SO3
was stable and
remained unaltered when it was irradiated with 360-kJ
visible light (Figure 4B, iv). Thus, oxidation of 6-TG to
G
SO2
in a Type II photochemical reaction can be
recapitulated using Rose Bengal and visible light as a
source of
1
O
2
.G
SO3
, in which the S atom is in the
highest oxidation state, is refractory to further oxidation
by
1
O
2
.
Direct chemical oxidation of 6-TG also proceeded via
a reactive G
SO2
intermediate to the ultimate product,
G
SO3
, which resisted further oxidation. When 6-TG was
treated under mild oxidizing conditions, G
SO2
was formed
as a stable product. Thus, oxidation with I
2
is the synthetic
route for G
SO2
(15) (Figure 1A, iv). Under more stringent
conditions, G
SO2
was susceptible to oxidation and the
pure compound was converted to G
SO3
in the presence
of Fe
2þ
in the form of Fe(NH
4
)
2
SO
4
in a concentration-
dependent manner (Figure 4C). These findings are consis-
tent with the favourable oxidation of 6-TG to G
SO2
as the
first stable reaction product. G
SO2
is further oxidized to
G
SO3
in a reaction that requires more rigorous conditions.
G
SO3
is refractory to further oxidation.
Ascorbate protects 6-TG against UVA-mediated oxidation
Ascorbate (vitamin C, Vc), an acknowledged antioxidant,
prevented the UVA-mediated destruction of 6-TG.
Solutions of 6-TG (0.1 mM) and ascorbate were UVA
irradiated and the loss of A
342
was monitored spectro-
photometrically. In the absence of ascorbate, a dose
of 11 kJ/m
2
UVA destroyed >80% of the 6-TG
(Figure 5A). A 12-fold molar excess of ascorbate during
irradiation conferred significant protection and >80% and
>30% of the 6-TG remained unchanged after UVA doses
of 11 and 23 kJ/m
2
, respectively (Figure 5A). Higher
ascorbate concentrations were even more protective and
approached completion at Vc:6-TG ratios of 25, at
which <20% of the 6-TG was destroyed by 30 kJ/m
2
UVA. Experiments conducted in parallel confirmed that
neither G
SO2
nor G
SO3
reacted with ascorbate (data not
shown). Ascorbate only protected against 6-TG oxidation
Figure 4. Chemical oxidation of 6-TG, G
SO2
and G
SO3
.(A) Rose
Bengal and light treatment of 6-TG. Aqueous 6-TG (0.1 mM) was
irradiated with visible light (200 W) at a dose of 360 kJ in the
presence of 0.5 mM Rose Bengal. Products were separated by HPLC
System 1. The relevant part of the absorbance (300 nm) trace is shown.
(B) Rose Bengal and light treatment of G
SO2
and G
SO3
. Aqueous G
SO2
(upper panels) or G
SO3
(lowest panel) (both 0.1 mM) were irradiated
with visible light for the indicated times in the presence of 0.5 mM Rose
Bengal. Products were separated by HPLC System 3. (C) Oxidation of
G
SO2
. Aqueous G
SO2
(0.1 mM) was incubated with the indicated con-
centrations of Fe(NH
4
)
2
SO
4
, and products analysed immediately by
HPLC System 3.
Table 1. 6-TG/UVA photoproducts in genomic DNA
UVA
(kJ/m
2
)
Measured
6-TGdR
(pmole)
Total
dG
SO2
(pmole)
Measured
dG
SO3
(pmole)
Estimated
dG
(pmole)
Recovery
(%)
0 510 0 0 0 100
5 282 158 30 7 94
15 158 230 53 26 92
45 65 271 78 54 92
DNA containing 6-TG (2% of guanine) was irradiated with UVA, and
digested to nucleosides which were separated and quantified. Data are
corrected for destruction of dG
SO2
as in the legend to Figure 3.
Nucleic Acids Research, 2010, Vol. 38, No. 6 1837
if it was present during the irradiation. When a 100-fold
excess of ascorbate was added to 6-TG that had been
previously irradiated with 21 kJ/m
2
, there was no change
in the amounts of photoproducts and 6-TG was not
regenerated (data not shown).
Ascorbate also prevented oxidation of 6-TG in an
oligodeoxynucleotide. A 10- or 20-fold molar excess of
ascorbate reduced by 2-fold the rate of UVA-induced
conversion from 6-TG to G
SO3
as monitored by
spectrophotofluorimetry (Figure 5B).
Reversal of 6-TG oxidation by Na
2
S
Na
2
S converts G
SO3
to 6-TG in a slow reaction that takes
several days at RT (6). When G
SO2
(0.1 mM aqueous
solution) was combined with Na
2
S at RT and the
mixture analysed immediately by HPLC, there was a sig-
nificant Na
2
S concentration-dependent conversion of
G
SO2
to 6-TG (Figure 6). A 20-fold excess of Na
2
S, con-
verted 90% of the input G
SO2
to 6-TG. This rapid and
efficient reaction is in contrast to the slow generation of
6-TG from G
SO3
.
DISCUSSION
We previously reported that 6-TG is labile to UVA irra-
diation both as a free base and when incorporated into
DNA (5,6). Exposure of 6-TG to UVA in the presence of
oxygen causes the formation of
1
O
2
. 6-TG is particularly
vulnerable to oxidation and is easily oxidized by Rose
Bengal plus visible light, an acknowledged
1
O
2
source.
The highly fluorescent DNA G
SO3
was previously
identified as a significant product of
1
O
2
-mediated oxida-
tion (5,6). 6-TG has a low oxidation potential and under
conditions in which normal DNA bases are invulnerable,
6-TG is quantitatively oxidized to G
SO3
by treatment with
the mild oxidizing agent MMPP. We proposed that,
on UVA exposure, G
SO3
was formed via the partially
oxidized intermediates guanine sulfenate (G
SO
) and
G
SO2
, both of which are reported to be unstable (16).
Figure 7 summarizes these reactions. In this report, we
identify G
SO2
as a major stable photoproduct of the free
6-TG base, its 20-deoxyribonucleoside, and of 6-TG in
DNA. Under mild conditions, G
SO2
is also the major
stable product of MMPP oxidation of 6-TG. The initial
oxidation of 6-TG to G
SO2
is more favourable than the
subsequent oxidation to G
SO3
.G
SO3
itself is refractory
to further oxidation. UVA irradiation of 6-TG or its
20-deoxyribonucleoside also generated small amounts
of G or dG. We did not observe any photoproduct,
Figure 5. Ascorbate protects against oxidation. (A) Protection of 6-TG
against photochemical destruction. The 6-TG (0.1 mM in aqueous
solution) was irradiated with the doses shown in the presence of differ-
ent concentrations of ascorbate. Photochemical destruction of 6-TG
was monitored by the reduction in A
342
spectrophotometrically. (open
circle) no ascorbate (filled circle) ascorbate:6-TG 3: 1 (filled square)
12: 1 (open square) 25: 1 (filled triangle) 100: 1. (B) Protection of
DNA 6-TG. A 11-mer oligonucleotide (1 mM) (CAGXAATTCGC
where X¼6-TG) was UVA irradiated in the presence of ascorbate as
indicated; 0 mM ascorbate (filled square), 10 mM (open circle) or 20 mM
(filled circle). Conversion of 6-TG to G
SO3
in the intact oligonucleotide
was monitored fluorimetrically (l
ex
320 nm; l
em
410 nm).
Figure 6. Reversion of G
SO2
by Na
2
S. Aqueous G
SO2
(0.1 mM) was
mixed with Na
2
S at RT to the final concentration indicated and the
sample was immediately analysed by HPLC System 3. Products were
detected by A
320 nm
. The known position of elution of 6-TG is shown
arrowed.
1838 Nucleic Acids Research, 2010, Vol. 38, No. 6
either from the free base or from DNA 6-TG, with
properties expected of guanine sulfenate (G
SO
). This
likely intermediate is possibly too unstable to withstand
our analysis procedure. In a description of the properties
of sulfenate, Abraham et al. (16) reported that it was not
possible to isolate free sulphenic acids, and instead
purified the silver salt of purine-6-sulfenate from which
purine sulphenic acid could be released by acidification.
The free acid had a relatively short half-life in aqueous
solution, particularly at neutral pH. Under acidic condi-
tions, purine-6-sulphenic acid was shown to break down
to 6-thiopurine and hypoxanthine via purine-6-sulphenic
acid. In the case of G
SO
, the analogous products would be
6-TG and G produced via G
SO2
.
This skin of patients taking thiopurines contains DNA
6-TG and is selectively hypersensitive to erythema induc-
tion by UVA (5,12). This is consistent with the formation
of replication- and transcription-blocking DNA lesions
(17). UVA treatment of cells containing DNA 6-TG
generates
1
O
2
in DNA and this is also associated with
severe inhibition of transcription and replication (6,10).
The formation of replication- and transcription-blocking
DNA lesions can be demonstrated in biochemical assays
using purified enzymes and UVA- or MMPP-treated
synthetic DNA substrates containing 6-TG. One aim of
this study was to characterize the 6-TG photoproducts in
DNA. In general, the DNA photoproducts qualitatively
and quantitatively mirrored those formed by irradiation of
free 6-TG or 6-TGdR in solution. The major exception is
formation of the G-S-G dimer. This would be unlikely
in DNA with a relatively low level of 6-TG substitution.
We confirmed the formation of G
SO3
and showed further
that G
SO2
is the major stable DNA photoproduct. Since
together these two lesions account for >90% of the
destroyed DNA 6-TG, at least at lower doses (45 kJ/m
2
),
they are likely to be the ones with the biggest impact on
biological processes.
In biochemical assays of DNA replication and tran-
scription using template oligos containing a single 6-TG,
the inhibition caused by a quantitative destruction of
template 6-TG by UVA irradiation or by MMPP are
closely similar (6,10). Our measurements reported here
indicate that in the former case, >80% of the photo-
products will be G
SO2
, whereas MMPP treatment results
in 100% conversion to G
SO3
. The similar effects of these
DNA lesions on DNA and RNA polymerases indicated
that DNA G
SO2
and G
SO3
, which contain large, negatively
charged substituent groups, are both very effective inhib-
itors of these enzymes.
The generation of damaging ROS is one of the inescap-
able hazards of aerobic metabolism and oxidation is a
constant threat to DNA. Cellular antioxidant defences
protect key cellular macromolecules from damage by
normal levels of ROS. In addition to being a photo-
chemical source of
1
O
2
, DNA 6-TG is an important
target for damage by cellular ROS, including H
2
O
2
(18).
We observed that ascorbate, an acknowledged dietary
antioxidant and a significant intracellular consumer of
1
O
2
, protected 6-TG and DNA 6-TG against photo-
oxidation. These findings suggest that antioxidant
defences may protect thiopurine-treated patients by pre-
venting the oxidation of DNA 6-TG. Additional protec-
tion might arise via reversion of the major photoproduct,
G
SO2
, back to 6-TG. Although G
SO3
is stable and its rever-
sion requires quite stringent conditions, the reaction of
G
SO2
is relatively favourable. Conversion of this poten-
tially replication- and transcription-blocking DNA lesion
back to the relatively innocuous 6-TG would ameliorate
the UVA-mediated biological effects of DNA 6-TG.
Overall, the oxidation of 6-TG via the reactive intermedi-
ates guanine sulfenate (G
SO
) and G
SO2
, and the persistence
of the relatively reactive G
SO2
is, however, likely to be
hazardous. Both G
SO
and G
SO2
are much more reactive
towards nucleophiles than G
SO3
the final oxidation
product. Their reactivity raises the possibility that inter-
mediate DNA G
SO
or accumulated G
SO2
, which forms in
the skin of UVA-exposed patients taking thiopurines, may
interact with cellular nucleophiles to form complex
addition products that might compromise cellular DNA
transactions and contribute to the extremely high
Figure 7. The 6-TG Oxidation: products and reactions. (A) Structures of 6-TG and oxidation products. (B) Reaction scheme for 6-TG.
(O) represents oxidizing treatment and, in particular the
1
O
2
that is generated by the interaction of 6-TG with UVA. The more favourable reactions
are shown with bold arrows.
Nucleic Acids Research, 2010, Vol. 38, No. 6 1839
incidence of sun-exposure-related skin cancer in this
patient group. The possible reactions of G
SO2
(and the
highly unstable guanine sulfenate, G
SO
) with cellular
reducing agents and nucleophiles are currently under
investigation.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We are grateful to members of the EPSRC Mass
Spectrometry Service Centre, University of Swansea for
recording some of the mass spectra.
FUNDING
Funding for open access charge: Cancer Research UK.
Conflict of interest statement. None declared.
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