Type II guanine oxidation photoinduced by the antibacterial fluoroquinolone rufloxacin in isolated DNA and in 2′-deoxyguanosine

Article abstract of DOI:10.1021/tx025530i

The role played by type I (radical) and type II (singlet oxygen) mechanisms in the Rufloxacin (RFX)-photoinduced production of 8-hydroxy-2′-deoxyguanosine in DNA has been evaluated. This fluoroquinolone drug has been shown to be able to photoinduce increased levels of some DNA base oxidation products, such as 8-OH-dGuo, that are indicative of mutagenic and carcinogenic events, with probable implications in aging processes. The relative weight of the two photosensitization mechanisms was obtained via determination of two different photoproducts of 2′-deoxyguanosine (dGuo), which are diagnostic of the two different pathways, namely, (4R*)- and (4S*)-4,8-dihydro-4-hydroxy-8-oxo-2′-deoxyguanosine and 2,2-diamino-4-[(2-deoxy-β-D-erythro-pentofuranosyl)amino]-2, 5-dihydrooxazol-5-one. The observed predominance of type II reaction is in agreement with the fact that the triplet state of RFX is able to transfer with high efficiency its energy to molecular oxygen, giving rise to singlet oxygen. Photophysical measurements suggest that hydrated electrons produced by Rufloxacin photoionization react with dGuo, Thd, and DNA, whereas these biomolecules quench the RFX triplet state with low efficiency. Static quenching of Rufloxacin fluorescence indicates an interaction of this drug both with DNA and with dGuo. On the basis of these experimental data, Rufloxacin photosensitization of DNA is proposed to occur by a type II mechanism.

Full text of DOI:10.1021/tx025530i

1142
Chem. Res. Toxicol. 2002, 15, 1142-1149
Typ e II Gu a n in e Oxid a tion P h otoin d u ced by th e
An tiba cter ia l F lu or oqu in olon e Ru floxa cin in Isola ted
DNA a n d in 2′-Deoxygu a n osin e
Alessandra Belvedere,† Francisco Bosca´,‡ Alfio Catalfo,† Maria C. Cuquerella,‡
Guido de Guidi,*,† and Miguel A. Miranda‡
Dipartimento di Scienze Chimiche, Universita` di Catania, Viale A. Doria 6, I-95125 Catania, Italy,
and Departamento de Qu´ımica/ Instituto de Tecnologı´a Quı´mica UPV-CSIC,
Universidad Polite´cnica de Valencia, Avenida de los Naranjos s/ n, 46022-Valencia, Spain
Received March 20, 2002
The role played by type I (radical) and type II (singlet oxygen) mechanisms in the Rufloxacin
(RFX)-photoinduced production of 8-hydroxy-2′-deoxyguanosine in DNA has been evaluated.
This fluoroquinolone drug has been shown to be able to photoinduce increased levels of some
DNA base oxidation products, such as 8-OH-dGuo, that are indicative of mutagenic and
carcinogenic events, with probable implications in aging processes. The relative weight of the
two photosensitization mechanisms was obtained via determination of two different photo-
products of 2′-deoxyguanosine (dGuo), which are diagnostic of the two different pathways,
namely, (4R*)- and (4S*)-4,8-dihydro-4-hydroxy-8-oxo-2′-deoxyguanosine and 2,2-diamino-4-
[(2-deoxy-â-D-erythro-pentofuranosyl)amino]-2,5-dihydrooxazol-5-one. The observed predomi-
nance of type II reaction is in agreement with the fact that the triplet state of RFX is able to
transfer with high efficiency its energy to molecular oxygen, giving rise to singlet oxygen.
Photophysical measurements suggest that hydrated electrons produced by Rufloxacin photo-
ionization react with dGuo, Thd, and DNA, whereas these biomolecules quench the RFX triplet
state with low efficiency. Static quenching of Rufloxacin fluorescence indicates an interaction
of this drug both with DNA and with dGuo. On the basis of these experimental data, Rufloxacin
photosensitization of DNA is proposed to occur by a type II mechanism.
Besides, due to the fact that chemical, biochemical, and
epidemiological studies regarding the evaluation of the
effect of oxidative stress on DNA in humans require the
development of noninvasive assays suitable for screening
purposes, the attention of researchers has been directed
toward the investigation of oxidized free bases and
nucleosides as DNA damage markers.
For example, according to recent studies, cigarette
smoking induces an increase of oxidative DNA damage
in lung tissue and peripheral leukocytes, which can be
quantified through the increase of 8-OH-dGuo levels (7-
9). In addition, a significant increase of 8-OH-dGuo
elimination in urines of smokers has been observed (10).
An increase of this oxidized base has been also found in
colorectal and uterine carcinoma cells, with respect to
nontumorous cells (11, 12).
In tr od u ction
The modification of purines and pyrimidines represents
one of the major classes of DNA damage induced by
reactive oxygen species (ROS),1 such as hydroxyl radicals,
superoxide anion, hydrogen peroxide, and singlet oxygen
(1).
Particularly, guanine has been shown to be the most
susceptible DNA target with regard to oxidation induced
by ROS, which are considered responsible for mutagenic
and carcinogenic events (2), with probable implications
in aging processes (3, 4). In this regard, studies carried
out on mice brain and heart have evidenced that protein
cross-linking, but more significantly the amount of 8-hy-
droxy-2′-deoxyguanosine (8-OH-dGuo), increases with age
ROS can also be produced following light absorption
by xenobiotic agents, which can act as phototoxic sensi-
tizers toward biological substrates such as DNA. In the
last years, scientific research is facing the problem of
human health damage caused by the action of sunlight
on a drug after its administration. Due to the ability of
sunlight to penetrate the skin layer to reach blood flux,
(4). In addition, the well-proven correlation between
oxidative damage to DNA and increased levels of 8-OH-
dGuo has permitted the utilization of this oxidized
nucleoside as a biomarker for DNA damage (5, 6).
1
Abbreviations: ct-DNA, calf thymus DNA; dGuo, 2′-deoxygua-
nosine; DAD, diode array detector; ECD, electrochemical detector; FLQ,
fluoroquinolone; NaOAc, sodium acetate trihydrate; NQS, 1,2-naph-
thoquinone sulfonic acid; 8-OH-dGuo, 8-hydroxy-2′-deoxyguanosine;
4-OH-8-oxodGuo, (4R*)- and (4S*)-4,8-dihydro-4-hydroxy-8-oxo-2′-deoxy-
guanosine; oxazolone, 2,2-diamino-4-[(2-deoxy-â-D-erythro-pentofura-
nosyl)amino]-2,5-dihydrooxazol-5-one; PB, phosphate buffer; PMT,
photomultiplier tube; RFX, Rufloxacin; ROS, reactive oxygen species.
* To whom correspondence should be addressed. E-mail: gdeguidi@
mbox.unict.it.
† Universita` di Catania.
‡ Universidad Polite´cnica de Valencia.
10.1021/tx025530i CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/08/2002

Type II Guanine Oxidation by Fluoroquinolone Rufloxacin
Chem. Res. Toxicol., Vol. 15, No. 9, 2002 1143
several drugs, both systemic and topic, could represent
a risk factor for oxidative damage toward DNA. Light
absorption by a drug molecule or metabolite can thus
induce photosensitization reactions able to provoke dam-
aging effects such as in vivo and in vitro phototoxicity,
photoallergy, photocarcinogenesis, etc. (13-17).
Moreover, an increasing amount of highly energetic
radiation is reaching the earth surface due to deep
alterations of the ozonosphere caused by environmental
pollution. Thus, the interest of the scientific community
toward this matter is dramatically increased, focusing
the attention of mass media and pharmaceutical com-
panies, which are interested in the reduction of these side
effects.
Recently, a remarkable increase in the utilization of
fluoroquinolones (FLQs) as a new class of antibacterial
drugs has been reported. These drugs are characterized
by a large and very efficient action spectrum and act
through inhibition of bacterial type II topoisomerase
(DNA girase and topoisomerase IV) (18).
In parallel, a number of reports have shown that these
drugs may be very efficient photosensitizers (19-21);
some of them are responsible for dangerous cutaneous
reactions (21-28), while others seem to be potential
photomutagenic and photocarcinogenic agents (29-32).
FLQ’s phototoxicity has been attributed to the forma-
tion of ROS, based on both in vitro (33-36) and in vivo
(20, 21) studies. Due to the side pharmacological target
of FLQs, DNA damage, and particularly guanine oxida-
tion, seems to be a very attractive matter of investigation
as regards FLQ-induced photosensitization.
Several authors have reported photoinduced guanine
oxidation associated with FLQs light exposure (35-39),
but at present, most of the information available in the
literature about the molecular mechanism is contradic-
tory or incomplete. For example, the use of ROS scav-
engers and quenchers was inconclusive as regards the
pathway (type I, radical or type II, singlet oxygen)
involved in the process (35). In addition, the ability of
FLQs to produce oxygen radical species and excited
molecular oxygen cannot be directly associated with their
phototoxicity (40-43). Indeed, it was reported that the
photooxidative potential does not necessarily correlate
with the photodegradation quantum yield (35-37). In
this context, a recent paper of Sauviago et al. has
proposed that the photosensitizing activity of some FLQs
to cellular DNA may involve an efficient energy transfer
to the double helix, in addition to the classical type I and
type II mechanisms (36).
the oxidative damage to DNA bases. This makes RFX
different from other FLQs, where the singlet oxygen route
is not efficient [i.e., Enoxacin (45) or Lomefloxacin (41)].
Also the photodegradation pathway is unusual: photo-
defluorination, which is common in the FLQ family (45-
48), does not occur to a significant extent. Further
peculiarities of RFX are the lack of influence of phosphate
on its photobehavior (49), and the predominating forma-
tion of N-demethylation product in the irradiation of RFX
in the presence of oxygen (50).
Exp er im en ta l P r oced u r es
Ch em ica ls. Rufloxacin hydrochloride was a gift from Bracco
(Milano, Italy) and Mediolanum (Milano Italy). 2′-Deoxygua-
nosine (dGuo), 8-hydroxy-2′-deoxyguanosine (8-OH-dGuo), nu-
clease P1, alkaline phosphatase, perinaphthenone, 1,2-naph-
thoquinone sulfonic acid (NQS), guanidine, TRIS, sodium citrate
dihydrate, sodium acetate trihydrate (NaOAc), ammonium
formate, and acetic acid were obtained from the Sigma Chemical
Co. (St. Louis, MO). Calf thymus DNA (ct-DNA) was purchased
from Pharmacia (Uppsala, Sweden). Phosphate buffer (PB)
consists of a 50 mM sodium phosphate solution adjusted to pH
7 with NaOH. Methanol, acetonitrile, and H2O were of HPLC
grade. All other chemicals were reagent grade.
Ir r a d ia tion Con d ition s. Irradiation was performed using
a Rayonet photochemical reactor equipped with eight black light
phosphor lamps emitting in the 310-390 nm range, with a
maximum at 350 nm. The fluence rate at the irradiation position
was about 800 µW/cm2.
The incident photon flux on the irradiated solutions in quartz
cuvettes (optical length, 1 cm), which is of the same order as
the solar fluence incident on skin, was in the range of 1 × 1016
quanta s-1. A “merry-go-round” irradiation apparatus was used
to ensure that all parallel samples received equal radiation. The
experimental procedures of irradiation and the light intensity
measurements have been described previously (51).
Tim e-Resolved Mea su r em en ts. A pulsed Nd:YAG SL404G-
10 Spectron Laser Systems was used for the excitation at 355
nm. The single pulses were ∼10 ns in duration, and the energy
was from 10 to 1 mJ /pulse. A pulsed Lo255 Oriel xenon lamp
was employed as the detecting light source. The laser flash
photolysis apparatus consisted of the pulsed laser, the Xe lamp,
a 77200 Oriel monochromator, and an Oriel photomultiplier tube
(PMT) system made up of a 77348 side-on PMT tube, 70680
PMT housing, and a 70705 PMT power supply. The oscilloscope
was a TDS-640A Tektronix. The output signal from the oscil-
loscope was transferred to a personal computer. If not otherwise
specified, all spectra were recorded in nitrogen-saturated solu-
tion.
Laser flash photolysis experiments with RFX (6 × 10-5 M)
were performed in the absence and in the presence of dGuo at
different concentrations (between 0.5 × 10-3 and 10 × 10-3 M)
in order to determine the bimolecular quenching rate constants
The aim of this work has been to gain insight into the
molecular mechanism of guanine moiety photooxidation
induced by the fluoroquinolone drug Rufloxacin, 9-fluoro-
2,3-dihydro-10-(4′-methyl-1′-piperazinyl)-7-oxo-7H-pyri-
do [1,2,3-de]-1,4-benzothiazine-6-carboxylic acid (RFX).
3
for reaction of RFX or the solvated electron with dGuo. These
constants were the slopes of the linear plots of the decay rate
3
constants of RFX (at 610 nm) or the solvated electron (at 730
nm) against dGuo concentrations. Similar experiments were
3
done with Thd and DNA to study the reactivity of RFX or the
solvated electron with these biomolecules.
Ultraviolet-visible absorption spectra were recorded on a
Beckman DU 650 spectrophotometer. Emission spectra were
obtained by a Spex Fluorolog-2 (model F-111).
Luminescence lifetimes were measured by an Edinburgh OB
900 time-correlated single-photon-counting spectrometer (N2
discharge, pulse width 2 ns). The Marquardt algorithm was used
to deconvolute the decay traces.
Ir r a d ia tion , P r ecip ita tion of DNA, a n d En zym a tic Hy-
d r olysis. ct-DNA was incubated at a concentration of 0.1 mg/
mL in 1720 µL of 50 µM RFX solution in PB and then irradiated.
After irradiation, DNA was precipitated by addition of 170 µL
Recently, the photosensitizing properties of this drug
have been studied (43, 44). The fact that a type II
mechanism mediates photodamage to membranes (43)
represents a good starting point for an investigation of

1144 Chem. Res. Toxicol., Vol. 15, No. 9, 2002
Belvedere et al.
of 3 M NaOAc by 5.1 mL of cold (-20 °C) absolute ethanol. After
vortexing, the mixture was stored at -20 °C for 2 h in the dark
and subsequently centrifuged for 15 min at 13000g at -10 °C.
The DNA pellet was separated from the solution, washed with
4 mL of cold 80% ethanol, and dried under a stream of nitrogen.
The DNA pellet was hydrolyzed with nuclease P1 to break 3′-
5′ sugar-phosphate bonds and with alkaline phosphatase to
remove the phosphate group; this procedure allows us to obtain
free nucleosides to be injected into HPLC. In particular, the
pellet was dissolved in 200 µL of 0.01 M TRIS-HCl (pH 7.00),
5.7 µL of 0.5 M NaOAc (pH 5.1), and 5 units of nuclease P1.
After vortexing and incubation at 37 °C for 60 min in the dark,
80 µL of 0.4 M TRIS-HCl (pH 7.5) and 3 units of alkaline
phosphatase were added to this solution. After 60 min, the
resulting hydrolysates were centrifuged for 20 min at 13000g
with an Eppendorf microfuge, using a 10 000 MW cutoff
Millipore filter in order to remove enzymes and other polymeric
residues such as undigested DNA. The filtrates were stored on
ice for no more than 4-6 h prior to analysis (see below). To avoid
artifacts, class A glassware was used (1).
solution was injected in HPLC. By monitoring the UV trace at
230 nm, the fractions relating to the 4S* and 4R* diastereoi-
somers, with retention times of 18 and 20 min, respectively,
were collected and lyophilized. The diastereomers were dissolved
in water and quantified using the molar absorption coefficient
of 5240 mol-1 cm-1 L-1 (55). Then the standard curve for 4S*-
4-OH-8-oxodGuo was constructed using the isolated standard
and correlating its concentration to the peak of the UV trace at
230 nm. The same procedure was applied for 4R*-4-OH-8-
oxodGuo.
Deter m in a tion of Typ e I d Gu o P h otop r od u cts. A more
sensitive method for the determination of type I photoproducts
is based on the reaction of guanidine produced by alkaline
hydrolysis of dGuo photoproducts with NQS to form a fluores-
cent product (56). In this case, 250 µL of the RFX-irradiated
solution with dGuo described above was treated after irradiation
with 250 µL of 1 N NaOH, for 15 min at 60 °C; then a 3 × 10-2
M aqueous solution of NQS (50 µL) was added and heated for 5
min at 60 °C. The resulting mixture was neutralized with 250
µL of 1 N HCl.
Deter m in a tion of 8-OH-d Gu o in DNA. Analysis of DNA
The quantitative evaluation of the fluorescent adduct, diag-
nostic for type I photoproducts of dGuo, and of the free
nucleoside was obtained by injecting the irradiated mixture in
a HPLC Kontron system model 420 equipped with a 432 UV
detector, an SFM fluorescent detector, and an SA 360 autosam-
pler. The separation was achieved on a LiChrosphere 100 RP-
18 column (5 µm packing, 4.6 mm i.d. × 25 cm) protected by a
LiChrosphere (5 µm packing) 4 × 4 mm guard column (Hewlett-
Packard), using as mobile phase 50 mM ammonium formate/
methanol (85:15 v/v). The UV detector wavelength was set at
260 nm. The excitation and emission wavelengths of the
fluorescence detector were respectively set at 355 and 405 nm.
The retention times for dGuo and the resulting fluorescent
product were respectively 6.7 and 8.3 min. The quantification
of dGuo and its type I photoproducts was achieved by standard
curves built with dGuo and guanidine, respectively.
hydrolysates was performed with
a Hewlett-Packard 1100
chromatograph equipped with an on-line diode array detector
(DAD) and an ESA Coulochem electrochemical detector, model
5100 (ECD) (ESA, Inc., Bedford, MA), with
a 5011 high-
sensitivity analytical cell (porous graphite) and a model 5020
guard cell, run in the screen mode.
For analysis, 20 µL of the filtrate was injected into a reverse-
phase Supelcosil LC-18-S (5 µm) 4.6 mm i.d. × 25 cm protected
by a Supelguard LC-18-S which was eluted with 12.5 mM
sodium citrate and 25 mM sodium acetate/acetic acid, pH 5.1,
with 7% methanol (v/v) at a rate of 1 mL/min. The UV traces
were monitored at 254 nm and at 293 nm, which represents
the absorption maxima of dGuo and 8-OH-dGuo, respectively.
The potentials of the electrochemical detector were set as
follows: guard cell, 430 mV; detector I, 120 mV; detector II, 380
mV; response time, 0.1 s; gain, 10 × 100. An 8-OH-dGuo
standard curve was constructed using 8-OH-dGuo standard and
correlating its concentration to the ECD response (52). In a
similar way, a dGuo standard curve was constructed using dGuo
standard and correlating its concentration with the integrated
UV absorbance at 254 nm. In particular, the area of the
experimental 8-OH-dGuo peak was used to calculate its con-
centration from the standard curve; the area of the experimental
dGuo peak from the same sample was used to calculate its
concentration from the standard dGuo curve. Results were
reported as (residues of 8-OH-dGuo vs residues dGuo) × 105
(53). Retention times for dGuo and 8-OH-dGuo were 18 and 27
min, respectively.
Resu lts a n d Discu ssion
P h otosen sitiza tion of DNA by Ru floxa cin (Gu a -
n in e Oxid a tion ). Photoinduced oxidative DNA damage
with formation of 8-OH-dGuo can occur either through
drug-derived radicals with attack to the position of dGuo
(8) or via singlet oxygen produced by energy transfer
processes (1). The RFX-sensitized photodegradation of the
dGuo moiety contained in the double helix was assessed
via HPLC equipped with DAD (Figure 1a), whereas
formation of 8-OH-dGuo was quantified through electro-
chemical detection (Figure 1b).
Figure 2 shows the photoinduced formation of 8-OH-
dGuo in a PB solution containing 0.16 mM ct-DNA and
50 µM RFX as a function of the irradiation time. As can
be noted, 8-OH-dGuo increases with increasing irradia-
tion time up to 30 min; further irradiation results in a
progressive decrease of this oxidized base. This can be
explained by considering the possibility of secondary
reactions, as the reactivity of 8-OH-dGuo toward singlet
oxygen is higher than that of the starting base (55). In
addition, about 80% of the drug is transformed within
20 min (data not shown). Thus, we cannot exclude that
RFX photoproducts could also be involved in 8-OH-dGuo
production and in its subsequent decrease.
The simultaneous analysis of both analytes on a single HPLC
injection provided excellent precision without the need for a
recovery standard or for rigorously quantitative sample han-
dling.
Deter m in a tion of Typ e II d Gu o P h otop r od u cts. dGuo
(1 mM) was irradiated with 3 mL of 50 µM RFX PB solution for
various periods of time. Formation of (4S*)-4,8-dihydro-4-
hydroxy-8-oxo-2′-deoxyguanosine and (4R*)-4,8-dihydro-4-hy-
droxy-8-oxo-2′-deoxyguanosine diastereomers (4-OH-8-oxodGuo)
was monitored with the HPLC chromatograph equipped with
DAD. Then 20 µL of the irradiated solution was injected in this
system, and the separation was achieved with a Supelcosil LC-
NH2 aminopropyl-substituted column (5 µm, 4.6 mm i.d. × 25
cm) protected by a Supelguard LC-NH2, by eluting in the
isocratic mode with a mixture of 25 mM ammonium formate/
acetonitrile (20:80 v/v) as the mobile phase at a rate of 1 mL/
min. The UV traces were monitored at 230 nm, which represents
the absorption maximum of the two diastereomers produced by
a type II mechanism, and at 254 nm for dGuo. A standard curve
for type II photoproducts was constructed using standards
prepared following the method of Stevenson with some modi-
fications (54): a 1 mM aqueous solution of dGuo was irradiated
in the presence of 55 µM perinaphthenone, and the irradiated
Besides, reaction of RFX with oxygen can trigger a
series of radical reactions with formation of superoxide
anion and hydroxyl radicals (57), which may also be
involved in dGuo oxidation. Moreover, the drug photo-
products can act as photosensitizers themselves, consid-
ering that the quinolone chromophore of these compounds
remains practically unaltered upon irradiation (43, 44).

Type II Guanine Oxidation by Fluoroquinolone Rufloxacin
Chem. Res. Toxicol., Vol. 15, No. 9, 2002 1145
Sch em e 1
to the formation of oxidation products in native DNA.
As a consequence, to elucidate the mechanism of dGuo
oxidation due to RFX photosensitization, a quantitative
determination of the different products formed from the
free nucleoside was performed (Scheme 1). These nucleo-
side photoproducts are the oxazolone derivative (2,2-
diamino-4-[(2-deoxy-â-D-erythro-pentofuranosyl)amino]-
2,5-dihydrooxazol-5-one), which arises via electron transfer
from dGuo to excited RFX (radical path) (59), and the
diastereomeric products 4R*- and 4S*-4-OH-8-oxodGuo
[which are diagnostic for a singlet oxygen attack to dGuo
(59)]. For this purpose, aliquots of 50 µM RFX PB
solutions were irradiated in the presence of 1 mM dGuo.
The irradiated samples were then divided into two
aliquots that were analyzed by HPLC for the parallel
evaluation of type I and type II photoproducts (Figure
3a and Figure 3b). Results reported in Table 1 show
clearly that up to a degree of dGuo transformation of
about 15% an almost quantitative conversion into the
type II product is observed. Under our experimental
conditions, the amount of photosensitized dGuo due to
UVA irradiation in the absence of RFX is negligible, even
after 90 min irradiation. These data are in agreement
with the significant quantum yield of singlet oxygen
production by RFX in buffered neutral solutions (44).
Em ission Stu d ies. Taking into account the relatively
short lifetimes of transient species involved in RFX
photodegradation (in particular, radicals and singlet
oxygen) and the rate constants of their reaction with
DNA (60), it appeared interesting to check the extent of
RFX-duplex interaction. Indeed, the photooxidation
pathways mediated by activated oxygen species are
favored, and energy/electron transfer processes between
excited RFX and the duplex can only occur if the two
molecules are quite close to each other (no more than 20
Å) (61). The absorption spectrum of RFX at pH 7.0, that
shows three bands centered at 244, 292, and 345 nm, was
not significantly affected by addition of dGuo. The
emission spectrum of RFX presents a large band with a
maximum at 470 nm. When increasing amounts of dGuo
in a concentration range between 1.1 and 7.7 mM were
added to 10 µM RFX, quenching of the drug emission (up
to a 25%) was observed (Figure 4). From the linear plot
of the ratio between RFX emission in the absence and in
the presence of quencher (Io/Iq) as a function of the
concentration of dGuo, a Stern-Volmer constant of 60.39
( 1.25 M-1 was calculated. Taking into account the RFX
fluorescence lifetime (4.8 ns) (44), a quenching constant
F igu r e 1. (a) HPLC chromatogram recorded by UV detection
of the sample obtained by precipitation and enzymatic hydroly-
sis of a PB solution of ct-DNA 0.16 mM photosensitized with
RFX 50 µM (irradiation time ) 30 min, a ) deoxycytidine, b )
methyl-deoxycytidine, c ) dGuo, d ) thymidine). (b) HPLC
chromatogram of the same sample recorded by electrochemical
detection.
F igu r e 2. Photoinduced formation of 8-OH-dGuo in a PB
solution containing ct-DNA 0.16 mM and RFX 50 µM as a
function of the irradiation time. The error bars represent the
standard deviation.
It is also worth mentioning that the maximum amount
of accumulated 8-OH-dGuo is about 3 × 10-12 mol, which
is only 1.5% of the total detected dGuo residues. This
value represents, however, a considerable amount, if
compared with the 8-OH-dGuo levels reached in “in vivo”
pathological processes (3-12, 58). Direct exposure to
UVA in the absence of photosensitizer results in less
8-OH-dGuo than that detected in the dark control.
F r ee d Gu o Nu cleosid e P h otooxid a tion . Formation
of 8-OH-dGuo in the double helix occurs through a
complex photosensitization mechanism involving a co-
operative action of both free radicals and singlet oxygen.
In addition, this process is favored by trace metals and
π-stacking of DNA bases (59). Hence, it is difficult to
evaluate the contribution of type I or type II pathways

1146 Chem. Res. Toxicol., Vol. 15, No. 9, 2002
Belvedere et al.
F igu r e 4. Changes of the emission spectrum of a 10 µM PB
solution of RFX in PB as a function of the dGuo concentration
(λexc 345 nm); [dGuo], mM: (a) ) 0, (b) ) 1.1, (c) ) 3.3, (d) )
7.7.
F igu r e 3. (a) HPLC chromatogram recorded by UV detection
of a 50 µM RFX PB solution irradiated for 50 min in the
presence of 1 mM dGuo for the quantification of type II
photoproducts. (b) HPLC chromatogram recorded by fluores-
cence detection of the same sample for quantification of type I
photoproducts.
F igu r e 5. Changes of the emission spectrum of a 10 µM
solution of RFX in PB as a function of ct-DNA concentration
(λexc 345 nm); [ct-DNA], mM: (a) ) 0, (b) ) 0.2, (c) ) 0.62, (d)
) 1.1.
Ta ble 1. F or m a tion of Typ e I a n d Typ e II P h otop r od u cts
in th e P r esen ce of Ru floxa cin w ith UVA Ir r a d ia tion in
Air -Sa tu r a ted Bu ffer Solu tion a
[type I products]
irradiation
time,
[photo-
detected by
[type II products]
detected by
degraded HPLC-fluorescence,
min
dGuo], µM
µM
HPLC-UV, µM
6
9
15
24
30
50 ( 3
70 ( 5
147 ( 11
150 ( 12
160 ( 13
1.9 ( 0.1
2.8 ( 0.1
4.4 ( 0.3
4.8 ( 0.3
4.9 ( 0.3
47 ( 4
65 ( 6
140 ( 13
145 ( 15
150 ( 16
a
The reaction was carried out upon irradiation of 50 µM RFX
in PB in the presence of 1 mM dGuo. The error represents the
standard deviation.
F igu r e 6. Transient difference absorption spectra observed at
100 ns after excitation of a nitrogen-saturated PB solution of
60 µM RFX with a 7 mJ 355 nm laser pulse: A in the absence
and B in the presence of 5 mM dGuo. A - B, difference between
both traces shows the typical absorption of the solvated electron.
of (1.26 ( 0.02) × 1010 M-1 s-1 was obtained.
Since the obtained value is higher than the diffusion
constant in water, a static quenching can be assumed.
This hypothesis is supported by the fact that the lifetime
of RFX fluorescence does not change in the presence of
different dGuo concentrations. This allows us also to
consider the Stern-Volmer value as an association
constant for the formation of the dGuo-RFX complex.
An analogous study was carried out with RFX emission
in the presence of DNA; in this case, up to 15% of
quenching was found under our experimental conditions
(Figure 5). The concentration range of DNA used was
between 0.2 and 1.1 mM. The calculated Stern-Volmer
constant was 162.3 ( 3.7 M-1, while the quenching
constant was (3.38 ( 0.05) × 1010 M-1 s-1. Again, the
lifetime of RFX emission did not change in the presence
of DNA, thus pointing to a static quenching.
La ser F la sh P h otolysis Stu d ies. Figure 6 shows the
time-resolved spectra obtained 100 ns after excitation of
60 µM RFX in PB solutions at 355 nm under nitrogen in
the presence and in the absence of 5 mM dGuo. The
difference between both traces shows the typical absorp-
tion of the solvated electron. Hence, the absorption
spectrum of the drug in degassed solution shows at least
two components: a fast decaying species at longer
wavelengths, which can be attributed to the hydrated
electron produced by RFX monophotonic photoionization
(50), and a second transient, peaking at 640 nm, with a
lifetime of 7 µs, assigned to the RFX triplet state (44).
Scheme 2 shows the most relevant processes occurring

Type II Guanine Oxidation by Fluoroquinolone Rufloxacin
Chem. Res. Toxicol., Vol. 15, No. 9, 2002 1147
Sch em e 2
reductive pathways end up with formation of superoxide
radical anion, either through direct trapping of the
electron by oxygen (reaction 4 in Scheme 2) or by reaction
of the radical anions of dGuo or Thd with oxygen, with
regeneration of the neutral species (not shown in the
scheme). In aqueous systems, the main reaction of
superoxide radical anion is dismutation into hydrogen
peroxide and molecular oxygen; neither superoxide nor
hydrogen peroxide seems to produce damage to the DNA
bases (62, 63).
The key steps of dGuo photooxidation via type I and
type II mechanisms are shown in Scheme 2, bottom.
Thus, dGuo and molecular oxygen compete for quenching
of RFX triplet; also the fact that the respective rate
constants (k15 and k10) differ by 2 orders of magnitude
justifies the predominance of the type II mechanism (see
Table 1 and corresponding comments above). Of course,
the absolute rates must also take into account the
concentrations of dGuo and oxygen, as well as the fact
that the reaction efficiency is not 100% (i.e., quenching
of 1O2 by dGuo does not necessarily lead to dGuo
oxidation products, and, on the other hand, back-electron-
transfer from RFX•- to dGuo•+ can greatly reduce the
3
efficiency of RFX quenching by the nucleoside).
In the case of thymidine, the analogous processes are
much slower, but type I mechanisms predominate (k17
>
k14). Thus, the overall reactivity of Thd is significantly
lower than that of dGuo. In principle, quenching of 3RFX
by Thd could also lead to triplet Thd as a result of energy
transfer, producing ultimately cyclobutane dimers. How-
ever, this is very unlikely in view of the relative triplet
energies, which make the process thermodynamically
disfavored by ca. 10 kcal/mol. As a matter of fact,
irradiation of 6 × 10-5 M RFX with 1 mM Thd under
aerated conditions (similar to those employed for dGuo)
did not produce any measurable degradation of the
nucleoside (data not shown).
The values found for DNA were consistently about 10
times lower than for the analogous reactions of dGuo.
Following the same reasoning as with the nucleoside, the
preferred reaction pathway for DNA would be a type II
mechanism. Taking into account the above comparison
between dGuo and Thd, guanines (rather than thymines)
would be reactive sites in DNA.
after excitation of aerated solutions of RFX in the
presence of DNA, dGuo, or Thd. In the present work, a
bimolecular quenching rate constant k5 ) 2.7 × 109 M-1
s-1 has been calculated for the quenching reaction of
solvated electrons by dGuo; this is in agreement with the
reported literature value (60). On the other hand, dGuo
does not seem to quench the triplet state of RFX with
significant efficiency, as indicated by the obtained bimo-
lecular quenching constant k15 ) 4.8 × 107 M-1 s-1
.
Time-resolved data were also obtained for the reaction
Con clu sion s
of 3RFX and hydrated electron with thymidine; this base
quenches electrons with a k6 value of 2.2 × 1010 M-1 s-1
,
On the basis of the overall results, a general picture
of RFX photosensitization of DNA and isolated nucleo-
sides can be proposed. As shown in Scheme 2, light
absorption leads to RFX in its singlet excited state, which
can undergo a photoionization process, giving hydrated
electrons and the RFX cation radical (RFX+•). Electrons
can be captured by suitable acceptors (RFX ground state,
dGuo, Thd, etc.), giving rise to radical anions. In air-
saturated solutions, superoxide radical anion and its
decay product hydroxyl radical would be formed. How-
ever, a more important pathway seems to involve RFX
triplet state, which would participate in energy transfer
to ground-state oxygen, with formation of singlet oxygen
[æ ) 0.32, (44)]. The type II photosensitization process
has been shown to be predominant as indicated by the
preferred formation of the diastereomeric 4R*- and 4S*-
4OH-8-oxo-dGuo, rather than the oxazolone derivative.
Thus, RFX is able to induce DNA photooxidation in
vitro with formation of modified guanine residues. Ex-
also in agreement with the literature (60). RFX triplet
quenching is not significant under our experimental
conditions (k17 < 107 M-1 s-1).
Similar studies performed in the presence of DNA point
to a less efficient quenching of both the RFX triplet (k16
< 107 M-1 s-1) and solvated electrons (k7 ca. 108 M-1 s-1);
this behavior is in agreement with the negligible effect
of DNA on the RFX photodegradation rate.
Some of the processes shown in Scheme 2 can be
included in a photoreductive pathway (reactions 4-7),
and do not result in oxidation of the DNA bases (60). The
oxidative pathways (reactions 12-17) are much more
important for DNA oxidation for the following reasons:
first, it has to be taken into account that the quantum
yield of photoionization (reaction 3 in Scheme 2) is 0.09
(50), while singlet oxygen formation (reaction 10 in
Scheme 2) is at least 3 times more efficient (quantum
yield 0.32) (44). Second, in the presence of oxygen, the

1148 Chem. Res. Toxicol., Vol. 15, No. 9, 2002
Belvedere et al.
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Ack n ow led gm en ts. Financial support from MURST
in the framework of the “Programmi di Ricerca di
Rilevante Interesse Nazionale” (project: Mechanisms of
photoinduced processes in organized systems), Universita`
degli Studi di Catania “Progetto Giovani Ricercatori”,
Consorzio Interuniversitario Nazionale “La Chimica per
l’Ambiente”, and Fundacio´n J ose y Ana Royo are grate-
fully acknowledged. We thank Prof. Sebastiano Campa-
gna (Dipartimento di Chimica Inorganica, Chimica Ana-
litica e Chimica Fisica, University of Messina, Italy) for
luminescence lifetime measurements.
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