Photochemistry and Photobiology, 2007, 83: 12261236

Photosensitizing Potential of Ciprofloxacin at Ambient Level

of UV Radiation
Neeraj Agrawal1, Ratan Singh Ray*1, Mohammad Farooq1, Aditya Bhushan Pant2 and
Rajendra Kumar Hans1
1
2

Photobiology Division, Industrial Toxicology Research Centre, M.G. Marg, Lucknow, India
In Vitro Toxicology, Industrial Toxicology Research Centre, M.G. Marg, Lucknow, India

Received 16 May 2006; Accepted 26 February 2007; DOI: 10.1562 2006-10-12-RA-1059

ABSTRACT
Ciprooxacin is a widely used uoroquinolone drug with broad
spectrum antibacterial activities. Clinical experience has shown
incidences of adverse effects related to skin, hepatic, central
nervous system, gastrointestinal and phototoxicity. India is a
tropical country and sunlight is abundant throughout the day. In
this scenario exposure to ambient levels of ultraviolet radiation
(UV-R) in sunlight may lead to harmful effects in ciprooxacin
users. Phototoxicity assessment of ciprooxacin was studied by
two mouse broblast cell lines L-929 and NIH-3T3. Generation of
reactive oxygen species (ROS) like singlet oxygen (1O2), superoxide anion radical (O2 )) and hydroxyl radical ( OH) was studied
under the exposure of ambient intensities of UV-A (1.14, 1.6 and
2.2 mW cm)2), UV-B (0.6, 0.9 and 1.2 mW cm)2) and sunlight
(60 min). The drug was generating 1O2, O2 ) and OH in a
concentration and dose-dependent manner. Sodium azide (NaN3)
and 1,4-diazabicyclo 2-2-2-octane (DABCO) inhibited the generation of 1O2. Superoxide dismutase (SOD) inhibited 9095%
O2 ) generation. The drug (540 lg mL)1) was responsible for
linoleic acid peroxidation. Quenching study of linoleic acid
peroxidation with SOD (25 and 50 U mL)1) conrms the
involvement of ROS in drug-induced lipid peroxidation. The
generation of OH radical was further conrmed by using specic
quenchers of OH such as mannitol (0.5 M) and sodium benzoate
(0.5 M). 2-deoxyguanosine (2-dGuO) assay and linoleic acid
peroxidation showed that ROS were mainly responsible for
ciprooxacin-sensitized photo-degradation of guanine base.
L-929 cell line showed 29%, 34% and 54% reduced cell viability
at higher drug concentration (300 lg mL)1) under UV-A, UV-B
and sunlight, respectively. 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) assay in NIH-3T3
cell line at higher drug concentration (300 lg mL)1) showed a
decrease in cell viability by 54%, 56% and 59% under UV-A,
UV-B and sunlight, respectively. Results of neutral red uptake
assay (NRU) in L-929 cell line were in accordance with MTT
assay. The NIH-3T3 cell line showed a higher photosensitizing
potential than L-929. The phototoxicity end point shows a
time- and concentration-dependent statistically signicant
(P < 0.001) damage. Ciprooxacin produced ROS by Type I
and Type II photodynamic reactions, interacted with nucleic acid
moiety and inhibited cell viability. Further, UV-induced photod

*Corresponding author email: rsray2001@rediffmail.com (Ratan Singh Ray)

 2007 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/07

1226

peroxidation of linoleic acid accorded the involvement of ROS in

the manifestation of drug phototoxicity. Appearance of ciprooxacin-induced phototoxicity at the ambient level of sunlight is
a real risk for the people of India and for those of other tropical
countries. We suggest that sunlight exposure should be avoided
(especially peak hours) during ciprooxacin treatment.

INTRODUCTION
Ciprooxacin is an important antimicrobial agent of the
uoroquinolone group. It is used for a wide range of infectious
diseases and is considered relatively safe (1). However,
uoroquinolones produce different adverse effects including
phototoxicity and photogenotoxicity (2,3). Some of the uoroquinolones used in chemotherapy have caused DNA photodamage and phototumorigenesis (4,5). Ciprooxacin has
shown incidence of undesirable adverse effects like gastrointestinal, skin, hepatic and central nervous system function
including phototoxicity (6). Gemioxacin and ciprooxacin
caused phototoxicity in healthy volunteers (7). India is a
tropical country and most of the human activities (agricultural,
commercial, sports, etc.) take place in bright sunlight, therefore, use of photosensitive drugs may lead to skin disorders
and immune suppression. Furthermore, human exposure to
UV-R in sunlight is increasing due to ozone depletion and
phototoxic effects of the drugs at the ambient level of UV-R in
sunlight are worth studying. Various drugs (antibiotics,
antimalarials, anti-inammatory agents, vitamins, etc.) with
UV-R are known to cause skin photosensitization via the
formation of reactive oxygen species (ROS) (8,9). Information
is needed regarding the phototoxicity and photocarcinogenecity of drugs for the safety of human beings (10).
UV-C and UV-B are absorbed largely within the epidermis
whereas around 50% of UV-A penetrates into the dermis (11).
Approximately 40% of the UV-B falling on the skin is
transmitted through the stratum corneum to viable epidermis
(12). Longer wavelengths are more penetrating (13). The
equivalent of the entire blood volume of an adult may pass
through the skin and is exposed within 20 min (14). Patients are
generally unaware that longer UV-R penetrates through
clouds, window glass and thin clothing and may produce
phototoxic responses (15). The phototoxic effects of UV-R are
related to the generation of ROS and induction of pyrimidine
dimmers (16,17). ROS have been implicated in many

Photochemistry and Photobiology, 2007, 83

pathological conditions including skin cancer and other
adverse effects caused by UV-R (18). UV-R caused dosedependent phototoxic effects on human erythrocytes (19).
Quinolone antibiotics, viz. peoxacin and ciprooxacin, caused
UVA-induced edema and immune suppression in mice (20).
Phototoxicity testing was recommended by the Organization for Economic Co-operation and Development, according
to which in vitro tests should be carried out before considering
any animal testing (21). The 3T3 NRU test was validated in
1998 for the phototoxicity assessment of chemicals including
uoroquinolones (22). In the present study, the photosensitizing potential of ciprooxacin was evaluated at the ambient
intensity of UV-R in sunlight by using two mouse broblast
cell lines L-929 and NIH-3T3 (23,24). The phototoxicity of
ciprooxacin was assessed by using 50 to 300 lg mL)1
concentrations of the drug. Chlorpromazine (5 lg mL)1) and
)1
L-histidine (100 lg mL ) were used as positive and negative
controls, respectively. The purpose of the study was to
evaluate the phototoxicity of ciprooxacin and its mechanism
of action at the ambient intensities of UV-R and sunlight by
using photochemical methods and a comparative analysis of
MTT and NRU assays by cell lines.

Photo-degradation of ciprooxacin. The drug was dissolved in Milli

Q deionized double distilled water and 10 lg mL)1 solution was
prepared. Drug solution was exposed to sunlight, aliquots were drawn
at intervals of 30 min, from 0 to 120 min. The photo-degradation
spectrum of the drug was recorded between 200 and 700 nm (Cintra 40
UV-visible spectrophotometer).
Photochemical assays. Determination of singlet oxygen (1O2). The
generation of 1O2 under aerobic conditions was measured in aqueous
solution as per the method of Ray et al. (25). RNO solution (0.35
0.4 10)5 M) was prepared in 0.01 M potassium phosphate buffer
(pH = 7.0) and L-histidine (10)2 M) was added as a selective acceptor
of 1O2. A 10 mL assay solution in a petri dish with or without the drug
was irradiated under UV-A (4.1047.92 J cm)2), UV-B (2.16
4.32 J cm)2) and sunlight (60 min). The production of 1O2 was
monitored by measuring the decrease in RNO absorbance at
440 nm. The generation of 1O2 was further substantiated by the
administration of two specic quenchers NaN3 (1 and 2 mM) and
DABCO (2 and 10 mM) (26,27).
Determination of superoxide anion radical (O2 )). The generation of
O2 ) was monitored by recording the photosensitized reduction of
NBT to nitro-blue diformazan (NBF) spectrophotometrically (28).
NBT solution (1.67 10)4 M) was prepared in 0.01 M sodium carbonate buffer (pH = 10). A 10 mL assay system containing the drug (1
25 lg mL)1) was irradiated with UV-A (1.3682.64 J cm)2), UV-B
(0.721.44 J cm)2) and sunlight (20 min). The production of NBF was
monitored by measuring the increase in the absorbance at 560 nm. The
generation of O2 ) was further conrmed by carrying out quenching
with SOD (25 U mL)1).
Determination of hydroxyl radical ( OH). The OH generation was
measured by ascorbic acid-iron-EDTA system proposed by Cohen
(29). The iron-catalyzed oxidation of ascorbic acid at 37C was used.
The standard reaction mixture contains 100 mM potassium phosphate
buffer pH 7.4, 167 lM iron-EDTA (1:2 mixture), 0.1 mM EDTA,
2 mM ascorbic acid and 33 mM dimethyl sulfoxide in a nal volume
of 3.0 mL and irradiated. Ascorbic acid was replaced by the drug
(25100 lg mL)1). After the completion of irradiation, 1.0 mL of
TCA (17.5%, wt vol) was added. The samples were then assayed for
formaldehyde formation by the method proposed by Nash (30).
Ammonium acetate acetylacetone reagent was prepared by 2.0 M
ammonium acetate, 0.05 M acetic acid and 0.02 M analytical grade
redistilled acetylacetone. Equal volumes of aliquot (1.5 mL) and
reagent (1.5 mL) were mixed and kept at 37C for 40 min. The
production of formaldehyde was monitored at 412 nm. Further the
quenching of OH was performed by adding mannitol (0.5 M) and
sodium benzoate (0.5 M) as specic quenchers.
Photodegradation of 2-deoxyguanosine (2-dGuo). To study the
radiation effects on guanine base of the nucleic acid, an indirect
method involving the photooxidative degradation of 2-dGuO was
used (31). 2-dGuO was prepared in 0.01 M sodium carbonate buffer
(pH = 10) and its optical density was adjusted between 1.2 and 1.4 at
260 nm. A 10 mL solution with or without the drug (2030 lg mL)1)
was irradiated under UV-A (4.10412.312 J cm)2), UV-B (2.16
6.48 J cm)2) and sunlight (60180 min). The degradation of 2-dGuO
was monitored by observing the decrease in absorbance at 260 nm.
Degradation of 2-dGuO was further substantiated by NaN3 (2 mM)
and DABCO (10 mM) (26,27).
Linoleic acid photoperoxidation. Linoleic acid solution was prepared
fresh in phosphate buffered saline (0.01 M, 0.9% NaCl, pH 7.2) using
0.05% Tween-20 as an emulsive agent. The solution containing 0.8 mM
linoleic acid and variable concentrations of the drug (040 lg mL)1)
were irradiated under ambient intensities of UV-A (1.368 J cm)2),
UV-B (0.72 J cm)2) and sunlight (20 min). Linoleic acid peroxidation
was measured by increase in absorbance at 233 nm (32). The
quenching of linoleic acid peroxidation was carried out by SOD (25
and 50 U mL)1).
Cytotoxicity assays. Cytotoxicity assays were performed by MTT
(23) and NRU methods (24).
Cell culture. The mouse broblast cell lines NIH-3T3 and L-929
were grown in DMEM F-12 HAM culture medium supplemented with
10% FBS and antibiotic-antimycotic solution (1.5%) at 5% CO2 and
95% relative humidity at 37C.
Phototoxicity assay. Cultured cells were seeded in 25 cm2 tissue
culture asks. The cells were trypsinized by trypsin-EDTA (0.25%)
after 36 h at 8090% conuency, mixed with equal volume of DMEM
d

MATERIALS AND METHODS

Chemicals and culture wares. N,N-dimethyl-p-nitrosoaniline (RNO),
SOD, nitro-blue tetrazolium (NBT), fetal bovine serum (FBS),
Dulbeccos modied eagles medium nutrient mixture F-12 HAM
(DMEM F-12 HAM), antibiotic and antimycotic solution, trypsin,
L-histidine,
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT), neutral red (NR), sodium azide (NaN3), DABCO,
tricarboxylic acid (TCA), sodium chloride (NaCl), ascorbic acid,
carbonate and phosphate buffers and 2-dGuO were procured from
Sigma (St Louis, MO). Linoleic acid and Tween-20 were obtained
from M.P. Biomedicals Inc. (Solon, OH) and Hanks Balanced Salt
Solution (HBSS) was purchased from Invitrogen Corporation.
Ciprooxacin was procured from Hi-Media India Ltd. Other reagents
and chemicals were procured from Hi-Media, Ranbaxy and Merck
India Ltd. Potassium ferricyanide [K3Fe(CN)6], ferrous sulfate,
ammonium acetate, acetylacetone and formaldehyde (HCHO) were
procured from M s Merck India and M s Qualigens, India. Pyrogen
free Milli Q double-distilled deionized water was used in the study.
All plastic wares including 48-well plates and 25 cm2 (polystyrene
coated) culture asks were purchased from Nunc. Cell lines were
procured from National Centre for Cell Sciences (Pune, India) and
since then it was maintained in our laboratory.
Radiation source and dosimetry. The UV-irradiation system comprised an array of 1.2 m long UV-R emitting tubes manufactured by
Vilber Lourmat (France). The intensity of emitted light was measured by
a microprocessor-controlled RMX-3 W radiometer (Vilber Lourmat)
equipped with calibrated UV-A, UV-B and UV-C detecting probes. The
spectral emission of UV-A source ranged from 320 to 400 nm with a
peak at 365 nm, whereas the spectral emission of UV-B source ranged
from 290 to 320 nm with a peak at 312 nm. The radiation dose was
measured in J cm)2. Intensities selected for irradiation were based on
dosimetry carried out at our laboratorys roof top between 12.00 P.M.
and 2.00 P.M. and were parallel to the ambient intensities of UV-A and
UV-B reaching in sunlight at Lucknow (26o45N latitude and 80o50E
longitude at 146 m above the mean sea level).
Radiation exposure. Radiation (UV-A and UV-B) exposure was
carried out at 25 2C in a radiation chamber. The samples were put
at a distance of 22.0 cm from the source of radiation. Glass petri dishes
(60 15 mm) were used for photochemical determination. Cell lines
were exposed in 48-well culture plates. Sunlight exposure was carried
out during clear sunny days between 12.00 P.M. and 2.00 P.M. at
25 2C. Petri dishes culture plates were kept on a platform
surrounded by ice packs (Polar Tech Industries, Genoa, IL) to prevent
increase in temperature.

1227

1228 Neeraj Agrawal et al.

F-12 HAM and collected in 50 mL sterilized plastic tubes. Cells were
washed thrice with HBSS. 2 104 cells were seeded per well in 48-well
plates. After incubation for 48 h, the medium was aspirated and cells
were washed with HBSS, prior to start of the experiment. A basal
control (cells only), dark control (drug-treated cells without light
exposure) and light control (cells exposed to light only) were also run
along with the experimental sets simultaneously under identical
conditions. Prior to UV exposure, drug-treated cells were incubated
for 30 min for the absorption of the drug by cells. Cells were then
exposed to the desired UV-R sunlight intensities followed by incubation for 30 min in a CO2 incubator. HBSS was replaced by DMEM F12 HAM and batches were processed further for cytotoxicity assays.
Cell viability assay by MTT: In brief, cells (2 104) were seeded per
well in 48-well plates and kept in the CO2 incubator for 48 h at 37C
prior to experiment for the proper attachment of the cells. The medium
was replaced by HBSS containing the drug for exposure purpose. At
the end of irradiation, HBSS was replaced by complete medium
containing MTT (20 lL of MTT [5 mg mL)1] with 200 lL complete
medium). The culture plates were kept in the CO2 incubator for 4 h.

1.2
1. Control (without exposure)
2. 30 min (exposure)
3. 60

4. 90

5. 120

Absorbance

1.0
0.8
0.6
0.4
0.2
0.0
225.0

275.0

325.0

375.0

425.0

Wavelength (nm)
Figure 1. Photo-degradation spectra of ciprooxacin at different time
intervals under sunlight exposure.

After incubation, the culture plates were washed twice with HBSS and
400 lL of DMSO was added to each well by pipetting up and down to
dissolve the content. The absorbance was recorded at 530 nm by using
multiwell microplate reader.
Neutral red uptake (NRU) assay. Briey, after irradiation, the test
drug was removed and washed with HBSS. The culture well plates
were allowed to incubate for 3 h in complete medium (DMEM F-12
HAM) containing NR dye (50 lg mL)1) followed by a quick wash
with xative (1% wt vol CaCl2; 0.5% vol vol formaldehyde) to
remove the unbounded dye. The accumulated dye was extracted with
50% ethanol containing 1% (vol vol) acetic acid and plates were kept
for 20 min on a shaker. The absorbance was recorded at 540 nm.
Statistical analysis. The mean and standard error deviations were
calculated. Students t-test was used to evaluate the signicance.

RESULTS
Ciprooxacin showed a strong absorption maxima (kmax) in
the UV-A (328 nm), UV-B (316 nm) and UV-C (276 nm)
regions (Fig. 1). Ciprooxacin exposed to sunlight (0120 min)
led to slight photo-degradation which increased with time. The
absence of isobestic points shows that no new photoproduct
was formed. As UV-C does not reach the earths surface,
phototoxicity study was carried out under UV-A, UV-B and
sunlight.
Table 1 shows the photochemical generation of 1O2 at
various concentrations of ciprooxacin when exposed to
different intensities of UV-A (1.14, 1.6 and 2.2), UV-B (0.6,
0.9 and 1.2 mW cm)2) and sunlight (60 min). Generation of
1
O2 was monitored at 10, 20, 40, 60, 80 and 100 lg mL)1
concentrations at different doses of UV-A (4.104
7.92 J cm)2), UV-B (2.164.32 J cm)2) and sunlight (60 min).
Ciprooxacin at 100 lg mL)1 generated the highest amount of
1
O2 under sunlight followed by UV-B and UV-A. The
formation of 1O2 was dependent on the drug concentration
and the dose of UV-R. Ciprooxacin (100 lg mL)1) did not

Table 1. Photochemical generation of 1O2 at various concentrations of ciprooxacin under the exposure of different intensities of UV-A (1.14, 1.6
and 2.2 mW cm)2), UV-B (0.6, 0.9 and 1.2 mW cm)2) and sunlight exposure (60 min).
Radiation exposure
UV-A
Drug conc.
(lg mL)1)

Intensity
(mW cm)2)

Dose
(J cm)2)

10
20
40
60
80
100
10
20
40
60
80
100
10
20
40
60
80
100

1.14

4.104

1.6

5.76

2.2

7.92

Values are mean of ve observations SD.

UV-B
DA at
440 nm
0.075
0.161
0.216
0.274
0.338
0.356
0.173
0.235
0.315
0.376
0.434
0.449
0.229
0.351
0.460
0.527
0.572
0.596

0.003
0.009
0.009
0.006
0.006
0.008
0.013
0.017
0.013
0.012
0.009
0.022
0.025
0.006
0.007
0.013
0.015
0.003

Intensity
(mW cm)2)

Dose
(J cm)2)

0.6

2.16

0.9

3.24

1.2

4.32

Sunlight
DA at
440 nm
0.226
0.341
0.484
0.560
0.588
0.638
0.190
0.322
0.503
0.593
0.623
0.649
0.272
0.411
0.532
0.630
0.685
0.746

0.004
0.006
0.006
0.039
0.033
0.024
0.005
0.030
0.017
0.045
0.007
0.002
0.024
0.014
0.014
0.004
0.020
0.007

Time
(min)
60

DA at
440 nm
0.204
0.378
0.501
0.608
0.707
0.791

0.022
0.003
0.005
0.007
0.009
0.002

Photochemistry and Photobiology, 2007, 83

produce 1O2 in the dark. No 1O2 generation was recorded
under UV-A, UV-B and sunlight without the drug. The lowest
yield of 1O2 was observed 10 lg mL)1 drug concentration
under UV-A (4.104 J cm)2) followed by sunlight (60 min) and
UV-B (2.16 J cm)2). Similarly, the highest intensity of UV-A
(2.2 mW cm)2 or 7.92 J cm)2), UV-B (1.2 mW cm)2 or
4.32 J cm)2) and sunlight (60 min) produced a higher amount
of 1O2. The 1O2 producing activity, based on the use of an
equivalent concentration of drug and UV-R was found in the
following ordersunlight>UV-B>UV-A.
In an attempt to evaluate the possible role of ROS in
ciprooxacin phototoxicity, quenchers (NaN3 and DABCO)
were used. Figure 2 shows the percent photochemical
quenching of 1O2 by NaN3 (1 and 2 mM) and DABCO (2
and 10 mM) at 100 lg mL)1 drug concentration under UV-A

1229

(4.104 J cm)2), UV-B (2.16 J cm)2) and sunlight (60 min).

NaN3 and DABCO showed the quenching from 39.1% to
58.6% and 7.6% to 62.5%, respectively. The highest quenching (58.6%) was observed with NaN3 (2 mM) under UV-B
and the lowest (7.6%) was observed with DABCO (2 mM)
under sunlight.
Table 2 shows the photochemical generation of O2 ) by
ciprooxacin at various concentrations (125 lg mL)1) under
different intensities of UV-A (1.14, 1.6 and 2.2 mW cm)2),
UV-B (0.6, 0.9 and 1.2 mW cm)2) and sunlight (20 min). The
highest yield of O2 ) was observed under sunlight (20 min) at
25 lg mL)1 drug concentration and the lowest yield by UV-A
at 1.14 mW cm)2 (1.368 J cm)2) by 1 lg mL)1 drug concentration. The order of O2 ) generating potential of ciprooxacin
at various concentrations was sunlight>UV-A>UV-B. UVA, UV-B and sunlight itself did not generate O2 ) alone.
Ciprooxacin between 1 and 25 lg mL)1 concentrations did
not produce O2 ) in the dark. The production of O2 ) was
concentration and dose dependent under UV-A, UV-B and
sunlight exposure. Additional evidence of the production of
O2 ) was obtained by examining the reduction in NBT and
concomitantly carrying out O2 ) quenching studies with SOD
(25 U mL)1). Quenching was 95.3% in sunlight, 92.8% in
UV-A and 92.3% in UV-B.
Table 3 shows the photodynamic degradation of 2-dGuO
at two concentrations of ciprooxacin (20 and 30 lg mL)1)
under various intensities of UV-A (1.14, 1.6 and
2.2 mW cm)2), UV-B (0.6, 0.9 and 1.2 mW cm)2) and sunlight
(60180 min). Photo-degradation was carried out at different
doses of UV-A (4.10423.76 J cm)2), UV-B (2.16
12.9 J cm)2) and sunlight (60180 min). The highest degradation of 2-dGuO was observed at 180 min under sunlight
followed by UV-B (12.96 J cm)2) and UV-A (23.76 J cm)2).
The 2-dGuO degradation was low at lower intensities and
d

UVA intensity (1.14 mW cm2)

% Quenching (1O2)

UVB intensity (0.6 mW cm2)

Sunlight (60 min)

70
60
50
40
30
20
10
0

1 mM

2 mM

2 mM

NaN3

10 mM
DABCO

Quenchers concentration

Figure 2. Percent photochemical quenching of 1O2 under UV-A

(4.104 J cm)2), UV-B (2.16 J cm)2) and sunlight exposure (60 min)
at 100 lg mL)1 ciprooxacin. Values are mean of three observations
SD.

Table 2. Photochemical generation of O2) by ciprooxacin at various concentrations (125 lg mL)1) under different intensities of UV-A (1.14, 1.6
and 2.2 mW cm)2), UV-B (0.6, 0.9 and 1.2 mW cm)2) and sunlight exposure (20 min).
Radiation exposure
UV-A
Drug conc.
(lg mL)1)

Intensity
(mW cm)2)

Dose
(J cm)2)

1
5
10
15
20
25
1
5
10
15
20
25
1
5
10
15
20
25

1.14

1.368

1.6

1.92

2.2

2.64

Values are mean of ve observations SD.

UV-B
DA at
560 nm
0.011
0.133
0.210
0.254
0.290
0.319
0.040
0.154
0.236
0.289
0.331
0.365
0.059
0.192
0.275
0.430
0.496
0.544

0.003
0.004
0.011
0.015
0.013
0.011
0.002
0.006
0.001
0.013
0.004
0.013
0.009
0.013
0.020
0.013
0.009
0.009

Intensity
(mW cm)2)

Dose
(J cm)2)

0.6

0.72

0.9

1.08

1.2

1.44

Sunlight
DA at
560 nm
0.019
0.110
0.173
0.207
0.238
0.261
0.035
0.154
0.239
0.302
0.348
0.438
0.076
0.208
0.323
0.412
0.492
0.525

0.008
0.004
0.006
0.007
0.007
0.009
0.001
0.005
0.016
0.017
0.013
0.006
0.003
0.005
0.014
0.008
0.012
0.003

Time
(min)
20

DA at
560 nm
0.185
0.292
0.420
0.519
0.584
0.662

0.009
0.011
0.024
0.008
0.018
0.018

1230 Neeraj Agrawal et al.

Table 3. Photodynamic degradation of 2-dGuO at 20 and 30 lg mL)1 concentrations of ciprooxacin under various intensities of UV-A (1.14, 1.6
and 2.2 mW cm)2), UV-B (0.6, 0.9 and 1.2 mW cm)2) and sunlight exposure (60, 120 and 180 min).
Radiation exposure
UV-A
Drug conc.
(lg mL)1)

Intensity
(mW cm)2)

Dose
(J cm)2)

20
30
20
30
20
30
20
30
20
30
20
30
20
30
20
30
20
30

1.14

4.104
8.208
12.312

1.6

5.76
11.52
17.28

2.2

7.92
15.84
23.76

UV-B
Percent
degradation
2.82
4.43
7.16
7.98
12.0
15.0
3.94
4.03
11.7
14.9
16.8
19.0
5.17
11.8
13.4
18.9
22.1
26.2

Sunlight

Intensity
(mW cm)2)

Dose
(J cm)2)

0.6

2.16

0.653
1.159
0.409
0.487
1.473
0.208
0.806
0.339
0.400
0.603
0.473
0.208
0.123
0.961
0.346
0.839
0.964
1.138

4.32
6.48
0.9

3.24
6.48
9.72

1.2

4.32
8.64
12.96

Percent
degradation
6.24
13.2
11.5
17.4
13.2
19.1
9.75
16.1
13.8
22.3
18.5
26.4
6.62
15.7
18.9
24.5
22.4
28.7

0.732
1.05
0.395
0.171
1.16
0.459
0.95
0.216
0.152
0.405
1.42
0.803
0.406
1.23
0.512
1.93
1.13
1.37

Time
(min)
60
120
180

Percent
degradation
16.1
21.5
24.9
27.6
26.4
33.5

0.173
0.300
0.115
1.21
1.60
1.36

% Quenching (2-dGuO)

Values are mean of ve observations SD.

120

UVA intensity (1.14 mW cm2)

UVB intensity (0.6 mW cm2)
Sunlight (60 min)

100
80
60
40
20
0
NaN3 (2 mM )

DABCO (10 mM )

Quenchers concentration
Figure 3. Percent quenching of photodynamic degradation of
2-dGuO by specic 1O2 quenchers under UV-A (4.194 J cm)2), UVB (2.16 J cm)2) and sunlight exposure (60 min). Values are mean of
three observations SD.

increased with higher intensities and doses. The rate of photodegradation of 2-dGuO was dose and concentration dependent in the following ordersunlight>UV-B>UV-A.
Figure 3 shows the percent quenching of photodynamic
degradation of 2-dGuO by 1O2 quenchers NaN3 (2 mM) and
DABCO (10 mM) under UV-A (4.194 J cm)2), UV-B
(2.16 J cm)2) and sunlight (60 min). NaN3 has greater quenching ability than DABCO. The highest quenching was observed
under UV-B (95.83%) in comparison with sunlight (68.5%)
and UV-A (69.9%). Quenching under UV-A and sunlight was
more or less the same.
Figure 4ad shows the photosensitizing effects of ciprooxacin at various concentrations from 50 to 300 lg mL)1 on the L929 cell line by recording percent cell viability. The phototoxicity
of ciprooxacin in the L-929 cell line was assessed using MTT
assay after 30 min of irradiation. Cytotoxicity was also observed

with different drug concentrations without irradiation. Chlorpromazine (5 lg mL)1) and L-histidine (100 lg mL)1) were
used as positive and negative photosensitizers, respectively. The
highest decrease in percent cell viability was observed at 90 min
while the lowest decrease was observed at 30 min exposure at all
the drug concentrations from 50 to 300 lg mL)1.
Figure 4a shows percent cell viability at different concentrations (50300 lg mL)1) of ciprooxacin under dark conditions. There was no signicant reduction in percent cell
viability at 50 and 100 lg mL)1 at 30, 60 and 90 min but at
higher concentrations (200 and 300 lg mL)1) a decrease in cell
viability was observed. 200 lg mL)1 drug concentration
showed 12%, 13% and 15% reduced cell viability in dark at
30, 60 and 90 min, respectively. Similarly, 300 lg mL)1 drug
concentration showed reduction in cell viability by 12%, 13%
and 16% at 30, 60 and 90 min, respectively.
Figure 4b shows the effect of drug concentrations (50
300 lg mL)1) on percent cell viability at 2.52, 5.04 and
7.56 J cm)2 doses of UV-A (1.4 mW cm)2).The drug concentrations at 200 and 300 lg mL)1 caused signicant
(P < 0.001) reduction in cell viability by 27% and 29%,
respectively, at 7.56 J cm)2 UV-A exposure.
Figure 4c shows the percent cell viability of the L-929 cell
line under UV-B (0.6 mW cm)2) exposure at 1.08, 2.16 and
3.245 J cm)2. At 1.08 J cm)2, cell viability was reduced. At
2.16 J cm)2, cell viability reduction was signicant
(P < 0.001) at 100, 200 and 300 lg mL)1 drug concentrations. The highest reduction (34%) was observed under
3.245 J cm)2 at 300 lg mL)1 drug concentration.
Figure 4d shows the photosensitizing potential of ciprooxacin under sunlight exposure from 30 to 90 min at different
concentrations. The highest reductions in cell viability were
42%, 48% and 54% at 300 lg mL)1 drug concentration
(P < 0.001) at 30, 60 and 90 min, respectively.

Photochemistry and Photobiology, 2007, 83

(a)

60

30

120
100
80
60
40
20
0

% Cell viability

% Cell viability

(a)

Dark

120
100
80
60
40
20
0

90

Dark

30

60

5.04

2.16

***
***
***

3.245

Exposure dose (J

5.04
7.59
Exposure dose (J cm2)

UV-B intensity (0.6 mW cm2)

1.08

cm2)

2.16
3.245
Exposure dose (J cm2)

Sunlight

(d)
100
80
60

Sunlight

(d)

120
% Cell viability

% Cell viability

% Cell viability

******
***

120
100
80
60
40
20
0

***
***
***

***
***
***
***

*** ***
***

40
20
0
30

60

90

120
100
80

******
*** ***

Drug (50 g

mL1)

Drug (200 g mL1)

***

***

***

******
***

60

***
***

40
20
0
30

Exposure dose (J cm2)

Chlorpromazine (5 g mL1)

***
***
***
***

2.52

(c)

***
*** ***
***

***
***

cm2)

UV-B intensity (0.6 mW cm2)

1.08

% Cell viability

7.59

Exposure dose (J

120
100
80
60
40
20
0

***
***

***

UV-A intensity (1.4 mW cm2)

120
100
80
60
40
20
0

% Cell viability

% Cell viability

2.52

(c)

(b)

UV-A intensity (1.4 mW cm2)

120
100
80
60
40
20
0

90

Exposure time (min)

Exposure time (min)

(b)

1231

60

90

Exposure dose (J cm2)

L-histidine (100 g mL1)

Drug (100 g mL1)

Chlorpromazine (5g mL1)

L-histidine (100 g mL1)

Drug (300 g mL1)

Drug (50 g mL1)

Drug (100 g mL1)

Figure 4. Photosensitizing effect of various concentrations of ciprooxacin on mouse broblast L-929 cell line as percent cell viability by
recording mitochondrial dehydrogenase activity (MTT assay) under
exposure of (a) dark, (b) UV-A, (c) UV-B and (d) sunlight.
Chlorpromazine (5 lg mL)1) and L-histidine (100 lg mL)1) were used
as positive and negative photosensitizers, respectively. Data expressed
are mean SE for four independent experiments. *P < 0.02,
***P < 0.001 as per Students t-test.

Figure 5ad shows the relative photosensitizing effect of

ciprooxacin at various concentrations (50300 lg mL)1) on
the L-929 cell line by recording NRU as percent cell viability.
L-929 was exposed to the drug for various radiation doses of
UV-A (1.4 mW cm)2) from 2.52 to 7.56 J cm)2, UV-B
(0.6 mW cm)2) from 1.08 to 3.245 J cm)2 and sunlight expo-

Drug (200 g

mL1)

Drug (300 g mL1)

Figure 5. Photosensitizing effect of ciprooxacin at various concentrations (50300 lg mL)1) on L-929 mouse broblast cell line by
recording NRU in lysosomes as percent cell viability. (a) Dark, (b)
UV-A, (c) UV-B and (d) sunlight exposure. Chlorpromazine
(5 lg mL)1) and L-histidine (100 lg mL)1) were used as positive and
negative photosensitizers, respectively. Values expressed are mean
SE for four independent experiments. ***P < 0.001 as per Students
t-test.

sure (3090 min). Chlorpromazine (5 lg mL)1) and L-histidine

(100 lg mL)1) were used as positive and negative photosensitizers, respectively. It is evident from the data that the decrease
in cell viability was concentration and dose dependent.

1232 Neeraj Agrawal et al.

% Cell viability

(a)

Dark

120
100
80
60
40
20
0
30

% Cell viability

***

% Cell viability

***

***
*** ***

2.52

(c)

90

60
Exposure time (min)
UV-A intensity (1.4 mW cm2)

(b) 120
100
80
60
40
20
0

*** ***

*** ***

***

5.04
7.56
Exposure dose (J cm2)

UV-B intensity (0.6 mW cm2)

120
100
80
60
40
20
0

***
***

1.08

***

2.16

***

***

3.245

Exposure dose (J cm2)

(d)
% Cell viability

Figure 5a shows percent cell viability of L-929 at different

drug concentrations (50300 lg mL)1) under dark conditions.
No signicant reduction in percent cell viability was observed.
Figure 5b shows the effect of different doses of UV-A (2.52,
5.04 and 7.56 J cm)2) on percent cell viability. At 2.52 J cm)2
exposure dose, a signicant (P < 0.001) reduction in cell
viability was observed at 200 and 300 lg mL)1 drug concentrations. Exposure to UV-A at 5.04 and 7.56 J cm)2 showed
signicantly (P < 0.001) reduced cell viability at all the drug
concentrations. The cell viabilities at 5.04 J cm)2 dose level
were 78.0% and 59.5% at 200 and 300 lg mL)1 drug
concentrations, respectively. Similarly, at 7.56 J cm)2 dose
level the cell viabilities were 54.1% and 25.2% at 200 and
300 lg mL)1 drug concentrations, respectively. The data
clearly show that the decrease in cell viability was dose and
concentration dependent.
Figure 5c shows the percent cell viability of the L-929 cell
line under UV-B exposure at 1.08, 2.16 and 3.245 J cm)2. No
signicant change in percent cell viability was observed.
Figure 5d shows the photosensitizing potential of ciprooxacin under sunlight exposure from 30 to 90 min. Dose- and
concentration-dependent signicant (P < 0.001) reduction in
cell viability was observed at all the drug concentrations.
Figure 6ad shows the relative photosensitizing response of
ciprooxacin on the NIH-3T3 cell line by using mito-chondrial
dehydrogenase activity as percent cell viability. Chlorpromazine (5 lg mL)1) and L-histidine (100 lg mL)1) were used as
positive and negative photosensitizers, respectively. NIH-3T3
was exposed with drug under UV-A (1.4 mW cm)2 intensity)
at 2.52, 5.04 and 7.56 J cm)2, UV-B (0.6 mW cm)2 intensity)
at 1.08, 2.16 and 3.24 J cm)2 and sunlight at 30, 60 and
90 min.
Figure 6a shows the percent cell viability at different
concentrations (50300 lg mL)1) of ciprooxacin under dark
conditions. There were mild reductions in percent cell viability
by 19%, 16% and 27% at 200 lg mL)1 and 25%, 23% and
30% at 300 lg mL)1 concentration under 30, 60 and 90 min
exposure, respectively. Lower concentrations of drug did not
have any impact on cell viability.
Figure 6b shows the effect of UV-A (1.4 mW cm)2 intensity) exposure from 2.52 to 7.56 J cm)2 on percent cell
viability. UV-A alone did not show any effect. A signicant
(P < 0.001) reduced cell viability was observed at all drug
concentrations under UV-A irradiation of 2.52, 5.04 and
7.56 J cm)2. The maximum reduced cell viability was observed
at 300 lg mL)1 concentration at all the doses of UV-A.
Figure 6c shows the percent cell viability under UV-B
exposure at 1.08 to 3.24 J cm)2. A signicant reduction
(P < 0.001) in percent cell viability was observed at all the
drug concentrations at 3.24 J cm)2. The highest reduction
(56%) in cell viability was observed at 300 lg mL)1. At
2.16 J cm)2 dose level, a signicant (P < 0.001) reduction in
percent cell viability was observed at 200 and 300 lg mL)1
drug concentrations. At 1.08 J cm)2 dose level a signicant
(P < 0.02) reduction in cell viability was observed only at the
highest concentration (300 lg mL)1).
Figure 6d shows the photosensitizing potential of ciprooxacin under sunlight exposure. A signicant reduction in
percent cell viability was observed at all drug concentrations
from 50 to 300 lg mL)1. The highest reduction (59%) was
observed at 300 lg mL)1 for 90 min exposure.

Sunlight
120
100
80
60

***
***

*** ***

***
*** *** ***

40

***
*** *** ***

20
0
30

60
Exposure dose (J cm2)

90

Chlorpromazine (5 g mL1)

L-histidine (100 g mL1)

Drug (50 g mL1)

Drug (100 g mL1)

Drug (200 g mL1)

Drug (300 g mL1)

Figure 6. Photosensitizing response of ciprooxacin on the NIH-3T3

cell line by using mitochondrial dehydrogenase activity (MTT assay) as
percent cell viability. (a) Dark, (b) UV-A, (c) UV-B and (d) sunlight.
Chlorpromazine (5 lg mL)1) and L-histidine (100 lg mL)1) were used
as positive and negative photosensitizers, respectively. Values
expressed are mean SE for four independent experiments.
***P < 0.001 as per Students t-test.

Figure 7 shows the photo-peroxidation of linoleic acid by

ciprooxacin under aerobic conditions in aqueous buffer (pH
7.2) solution by UV-A (1.14 mW cm)2) 1.368 J cm)2, UV-B
(0.6 mW cm)2) 0.72 J cm)2 and sunlight (20 min). Different
drug concentrations from 5 to 40 lg mL)1 were selected for

nmol mL1 of HCHO

UV-A = 1.14 mW cm2

UV-B = 0.6 mW cm2
Linoleic acid (0.8 mM)
0.9
" + 5 g mL1 drug
0.8
" + 10 "
0.7
0.6
" + 20 "
0.5
" + 40 "
0.4
0.3
0.2
0.1
0
UVB
UVA
(0.72 J cm2)
(1.368 J cm2)

35
30
25
20
15
10
5
0

25 g mL1

50 g mL1

UV-A
Sunlight
(20 min)

Radiation dose (J cm2)

Figure 7. Photoperoxidation of linoleic acid by ciprooxacin under
UV-A (1.368 J cm)2), UV-B (0.72 J cm)2) and sunlight exposure
(20 min). The formation of dienic hydroperoxides was followed by the
increase in absorption at 233 nm. Values presented are mean of four
observations SD.

exposure. Lipid peroxidation was higher under sunlight

followed by UV-B and UV-A. Lipid peroxidation was dose
and concentration dependent under all radiation doses.
Linoleic acid itself caused lipid peroxidation under UVR sunlight but higher yield was observed with different drug
concentrations.
Figure 8 shows the percent quenching of linoleic acid
peroxidation caused by ciprooxacin (20 lg mL)1) through
SOD (25 and 50 U mL)1) at UV-A (1.368 J cm)2), UV-B
(0.72 J cm)2) and sunlight (20 min). The percent quenching of
linoleic acid peroxidation by SOD (25 U mL)1) were 23.3%,
25.3% and 24.0% under UV-A, UV-B and sunlight, respectively. The percent quenching by SOD (50 U mL)1) were
51.6%, 34.9% and 43.3% under UV-A, UV-B and sunlight,
respectively. The highest quenching was observed under UV-A
and the lowest under UV-B while sunlight showed moderate
quenching.
Figure 9 shows the photochemical generation of OH at
various concentrations of ciprooxacin (25100 lg mL)1)
under UV-A (4.32 J cm)2), UV-B (2.16 J cm)2) and sunlight
(60 min). The drug-induced OH generation was concentration
dependent. The maximum generation of OH was observed
under sunlight followed by UV-A and UV-B.
d

75 g mL1

UV-B
Exposure

1233

100 g mL1

Sunlight

Figure 9. Photochemical generation of OH at various concentrations

of ciprooxacin under UV-A (4.32 J cm)2), UV-B (2.16 J cm)2) and
sunlight exposure (60 min). Values presented are mean of three
observations SD.

% Quenching

OD at 233 nm

Photochemistry and Photobiology, 2007, 83

100
90
80
70
60
50
40
30
20
10
0
Mannitol (0.5 M)

Sodium benzoate (0.5 M)

Quenchers concentration
Figure 10. Percent quenching of OH at 100 lg mL)1 ciprooxacin by
mannitol and sodium benzoate under UV-A (4.32 J cm)2). Values
presented are mean of three observations SD.
d

Figure 10 shows the percent quenching of OH under UV-A

(4.32 J cm)2) at 100 lg mL)1 ciprooxacin by mannitol
(0.5 M) and sodium benzoate (0.5 M). Both the quenchers
caused 20% to 30% quenching of OH. Higher quenching was
observed by sodium benzoate than by mannitol.
d

% Quenching by SOD

100
90
80
70
60
50
40
30
20
10
0

25 units mL1
50 units mL1

UV-A

UV-B

Sunlight

Figure 8. Photochemical quenching of linoleic acid (0.8 mM) peroxidation by SOD (25 and 50 U mL)1) under UV-A (1.368 J cm)2),
UV-B (0.72 J cm)2) and sunlight exposure (20 min). Values presented
are mean of four observations SD.

DISCUSSION
Ciprooxacin is a quinolone carboxylic acid derivative and has
gram-negative and gram-positive bactericidal activities. It is
rapidly absorbed in the body. Three major metabolites,
desethylene-ciprooxacin (metabolite M1), sulfo-ciprooxacin
(metabolite M2) and oxo-ciprooxacin (metabolite M3) are
formed in the body uids (33). Its irradiation under sunlight
leads to slight photodegradation. The absence of isobestic
points shows that no new photoproduct was formed under
sunlight irradiation. This study suggests that ROS generation
and DNA damage by photoilluminated ciprooxacin were
mainly responsible for phototoxicity on L-929 and NIH-3T3
cell lines. Perhaps, this is the rst report demonstrating the
phototoxicity of ciprooxacin and its mechanism under
ambient levels of UV-A, UV-B and sunlight. Sunlight is a
broad spectrum of different wavelengths containing UV-A,
UV-B and many other radiations, therefore the effect of
sunlight may be cumulative and higher. Earlier studies were
performed at higher doses of UV-R (34,35). The uoroquinolones absorb light in the UV-A wavelength from 320 to 330 nm

1234 Neeraj Agrawal et al.

and produce ROS such as 1O2, O2 ), H2O2 and OH. Thus the
photodynamic generation of ROS may be the basis of
phototoxicity of quinolones in human beings and animals
(36). Our study indicates that the commonly used ciprooxacin
was generating 1O2, O2) and OH through photosensitized
mechanisms, in which the photoexcited drug (triplet state)
transfers its energy to O2 and generates energy-rich 1O2 (via
Type II photodynamic reaction), an active oxidizing agent.
Another oxygen-dependent reaction involves the photoreaction of an electronically active photosensitizer, releases the
electron or by hydrogen transfer from a compound to O2 and
produces O2 ) and OH (via Type I photodynamic reaction).
Quenching studies with specic quenchers of 1O2 and OH
showed that 1O2 and OH were predominant in photodegradation of 2-dGuO. Sodium azide and DABCO were
able to control the generation of 1O2 and mannitol, and
sodium benzoate was able to quench OH. Quenching with
SOD conrms the generation of O2 ) by ciprooxacin. Rosen
et al. (37) have worked out the mechanism of ROS generation
under UV-A-exposed lomeoxacin and ciprooxacin at the
exposure of 20 J cm)2 and found that the phototoxicity of
these drugs was mediated through photodynamic reactions. In
our study ambient levels of UV-A and UV-B generated ROS
through photodynamic action. Sunlight (60 min) exposure also
produced similar effects which were not reported earlier. Our
earlier studies have shown that the ROS generation and
photodegradation of 2-dGuO are important factors in phototoxicity (25,28,38,39). Photosensitization appears to be the
main cause of phototoxicity of quinolones, particularly in
older patients with long-term use (40). Double-blind placebo
and skin phototesting potential in human beings were investigated by systemically administered uoroquinolones (41) but
these studies did not offer any mechanistic approach. Due to
the complexity of the biological system, it is difcult to
demonstrate clearly the specic role of ROS in the manifestation of phototoxicity in in vivo studies. It is clear that 1O2 is
an important ROS in DNA damage under in vitro conditions
(42,43). In photosensitization reactions 1O2, O2 ) and OH are
formed which on interconversion lead to the generation of
H2O2 (9). These reactive forms of oxygen may be responsible
for linoleic acid peroxidation (32).
2-dGuO was used to assess the nuclear damage caused by
sensitivity of the guanine base to UV-R sunlight. Signicant
photo-degradation of 2-dGuO was observed by the photosensitized ciprooxacin. Lomeoxacin and ciprooxacin
showed the photodynamic ability of 8-oxo, 7-8-dihydro-2deoxyguanosin formation in adult rat liver-18 cells under UVA exposure. Our observation is in accordance with that of
Rosen et al. (37). Commonly used antibiotics such as cephaloridine, cephalexin and cephradine were found to generate
signicant amounts of 1O2 and caused photo-degradation of
2-dGuO under UV-R (25). Antibiotics like lomeoxacin and
enoxacin generated 1O2 and O2 ) under UV-R and sunlight,
caused lipid peroxidation in human blood and photodegraded
2-dGuO in vitro (39). The highly photoreactive lomeoxacin
and BAYy3118 have caused photo-induced DNA strand
breaks (3). Photoirradiation of neuroleptic drugs with cells
released lactate dehydrogenase and caused the loss of mitochondrial NADH dehydrogenase activity, indicating that
plasma membrane and mitochondria were the targets of
phototoxicity (35).
d

UV-R-induced impairment of proteasome function has

been investigated in human keratinocyte culture (44). UV-R
caused delayed mutations and chromosomal instability in
broblasts (45). Ciprooxacin signicantly reduced the cell
viability of the NIH-3T3 cell line compared with the L-929
cell line. The MTT assay results were comparatively
signicant in the NIH-3T3 cell line. A high reduction of
cell viability in the NIH-3T3 cell line under sunlight
exposure had demonstrated that the damaging potential of
sunlight was more lethal compared with UV-A and UV-B.
The selection of different intensities of UV-A and UV-B
radiation prove our viewpoint that higher intensities of
UV-A and UV-B radiation in sunlight (in view of ozone
depletion) would be more deleterious. Therefore, the measurement of ambient intensity and the total dose is an
important factor for phototoxicity assessment.
The results show that MTT and NRU assays are high quality
and suitable for identifying phototoxicity. Lomeoxacin-treated normal human skin cells showed UV-A-induced DNA
strand breaks and pyrimidine dimmers in genomic DNA. The
phototoxicity of the drug can be drawn by different mechanisms. The photobiological impact of the drug varies according
to cell types (46). The effect on L-929 and NIH-3T3 cell lines
point toward the involvement of ROS and DNA damage in
phototoxicity. It may suggest a link between the phototoxic and
photocarcinogenic potential of ciprooxacin. The phototoxicity of the drug on cell lines and lipid peroxidation of linoleic acid
suggest that the ROS might initiate cellular lipid peroxidation
and DNA damage which nally leads to cell death.
To examine the validity of this hypothesis, linoleic acid
photo-peroxidation was used as a model system (47,48). The
ability of ciprooxacin to induce photodynamic lipid peroxidation and cell damage may be expected in vivo. SOD was
used as an antioxidant quencher for the protection quenching
of linoleic acid peroxidation. In an earlier study SOD was used
as a specic quencher of O2 ) (28). Percent quenching of
linoleic acid peroxidation by SOD accorded the involvement of
ROS in ciprooxacin phototoxicity. The generation of the OH
radical by ciprooxacin under sunlight and UVR is a
signicant indication of the reactivity of photosensitized
ciprooxacin toward nucleic acid bases. This was further
conrmed by the quenching of the OH radical by specic
quenchers (mannitol and sodium benzoate). The production of
ROS, photo-degradation of 2-dGuO and photodamage in cell
lines (in vitro) appeared to be the possible mechanisms of
ciprooxacin phototoxicity and photogenotoxicity (49).
In conclusion, the study suggests that ciprooxacin generates oxygen-dependent Type I and Type II photosensitizing
reactions, producing 1O2, O2 ) and OH which leads to photodegradation of 2-dGuO, linoleic acid peroxidation and cell
damage. The drug is generally used for a period of 1 week and
above for the treatment, so the use of drug and sunlight
exposure may be harmful and lead to phototoxic photogenotoxic effects. This advocates avoiding outdoor activities
(especially in peak hours) during ciprooxacin treatment.
d

AcknowledgementsThe authors are thankful to the Director, ITRC,

for his keen interest in the study and to Mr. B. D. Bhattacharji for
editing the manuscript. This study was nancially supported in part by
the Council of Scientic & Industrial Research, New Delhi, under
Network project (CMM-0018).

Photochemistry and Photobiology, 2007, 83

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