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means of the stroboscopic technique, which is a variation of the
boxcar technique. A hydrogen/nitrogen flash lamp (1.8-ns pulse
width) was used as excitation source. The kinetic traces were fitted
with monoexponential decay functions. Measurements were done
under aerated conditions at room temperature (25 1C) in cuvettes of
1-cm path length. The excitation wavelength used to register the
fluorescence lifetime was 320 nm. The fluorescence quantum yield
of quinine bisulfate in 1N H2SO4 (ϕF¼0.546) was used as standard.
Fluoroquinolone fluorescence quenching by DNA after excitation
at 355, 348, and 330 nm was performed using 10ꢁ4 M FQ buffered
aqueous solutions (10ꢁ3 M PB, pH ꢂ7.4). The DNA concentrations
were determined spectrophotometrically taking into account a
molar extinction coefficient ε258 nm¼6700 cmꢁ1 Mꢁ1 [22,26].
Eq. (1) was selected to determine the drug–DNA interactions from
fluorescence quenching data [27–31]:
Chart 1. Structure of LFX and its N-acetyl derivative ALFX.
binding between LFX and this nucleotide (LFX–dGMP) [21]. How-
ever, studies concerning the association of LFX with DNA have
revealed that an electron transfer reaction between the singlet
excited state of complexed LFX (1LFX…DNA) and DNA must also be
involved in the photodehalogenation because, despite the impor-
tant decrease in LFX emission, the efficiency of this process does
not change in the presence of increasing amounts of DNA [22].
Thereby, the literature findings suggest that 1LFX…DNA and 3LFX
might be candidates for covalent binding of LFX to DNA.
With this background, the main processes involving LFX photo-
degradation in the presence of 20-deoxyguanosine (dGuo) or DNA
were evaluated by performing emission studies, laser flash photo-
lysis, pulse radiolysis, and product analysis using ultraperformance
liquid chromatography with high-resolution mass spectrometry
detection (UPLC–HRMS). In this context, DNA photodamage was
assessed through the detection of single-strand breaks in plasmid
pBR322 and by examination of the UV–Vis absorption and fluor-
escence changes in DNA after its photosensitization with FQ and
subsequent separation by gel-filtration chromatography to inves-
tigate photobinding of LFX to DNA.
F0=F ¼ 1þKsv½Qꢃ;
ð1Þ
where F0 and F are the fluorescence intensities in the absence and
presence of the quencher, respectively; [Q] is the quencher con-
centration (DNA from 10ꢁ5 to 1.5 ꢀ 10ꢁ3 M in nucleotides); and Ksv
is the Stern–Volmer quenching constant.
Laser flash photolysis experiments
A pulsed Nd:YAG laser was used for the excitation at 355 nm.
The single pulses were ꢂ10 ns duration and the energy was from
10 to 1 mJ/pulse. A pulsed xenon lamp was employed as detecting
light source. The laser flash photolysis apparatus consisted of the
pulsed laser, the Xe lamp, a monochromator, and a photomulti-
plier made up of a tube, housing, and power supply. The output
Moreover, as it has been established that acetylation of the
piperazinyl ring of FQs produces changes in their photophysical
and/or photochemical behavior [17,23–25], some key experiments
were also performed using the lomefloxacin acetylated derivative
7-(4-acetyl-3-methyl-1-piperazinyl)-1-ethyl-6,8-difluoro-1,4-
dihydro-4-oxoquinoline-3-carboxylic acid (ALFX).
signal from the oscilloscope was transferred to
computer.
a personal
Aqueous solutions of 10ꢁ4 M (A)LFX were prepared in 10ꢁ3
M
NaHCO3 and the experiments registered under anaerobic condi-
tions bubbling N2O. Transient absorption spectra at different times
after the laser pulse were obtained for each sample in the presence
and the absence of DNA, paying special attention to intersystem
crossing quantum yield changes and to the generation of new
intermediates. The DNA concentrations ranged between 10ꢁ4 and
10ꢁ2 M in nucleotides.
The quenching experiments were carried out keeping the pH
constant at 7.4 throughout the experiment.
Rate constants of aryl cation quenching by biomolecules were
determined using the Stern–Volmer Eq. (2):
Materials and methods
General materials
Calf thymus DNA, ciprofloxacin (CFX), dGuo, flumequine (FM),
and LFX were commercial products obtained from Sigma–Aldrich,
whereas plasmid pBR322 was supplied by Roche and Sephadex
G-25 columns by GE Healthcare. Sodium phosphate buffer (PB)
and sodium bicarbonate buffer were prepared from reagent-grade
products using Milli-Q water; the pH of the solutions was
measured through a glass electrode and adjusted with NaOH to
pH 7.4. Other chemicals were of reagent grade and used as
received.
1=τ ¼ 1=τ0 þk½Qꢃ:
ð2Þ
Pulse radiolysis
The pulse radiolysis experiments were carried out with a
12-MeV Radiation Dynamics Ltd. (UK) 3-GHz electron linear
accelerator. We used a single-pulse mode with a pulse duration
from 0.22 to 2 μs and with a peak current of about 30 mA. The
accelerator is normally operated at 10 pulses per second but the
single-pulse mode is achieved by modifying the pulses to the gun
[32]. The detection system consisted of a Xe arc lamp and a pulsing
unit, high-radiance Kratos monochromator, and quartz optics.
Optical transmissions at various wavelengths selected with the
monochromator, bandwidths 10 nm, were observed as a function
of time before and after the radiation pulse using photoelectric
detection. The output of the photomultiplier (EMI 9558Q) was
displayed on a Tektronix TDS 380 digitizing oscilloscope. Data
processing was performed on a Dan PC using software developed
in-house. The sample cell, constructed from Spectrosil quartz, had
an optical path length of 25 mm [32].
The samples of FQs were prepared with various PB concentra-
tions starting from a stock solution of 300 mM PB adjusted to pH
7.4. ALFX was prepared as previously described from a solution of
LFX (300 mg, 0.9 mmol) in Ac2O (50 ml) that was refluxed for 7 h
[17]. The solution was cooled to room temperature and concen-
trated. Afterward, the residue was dissolved in water, neutralized
to pH ꢂ7.4, extracted with CH2Cl2, and concentrated to dryness.
Absorption and emission measurements
Ultraviolet spectra were recorded on a UV–Vis scanning spectro-
photometer (Cary 50). Fluorescence emission spectra were recorded
on a Photon Technology International (PTI) LPS-220B fluorimeter.
Lifetimes were measured with a time-resolved spectrometer (Time-
Master fluorescence lifetime spectrometer TM-2/2003) from PTI by