Production of ROS by photosensitized anthracene under sunlight and UV-R at ambient environmental intensities

Article abstract of DOI:10.1111/j.1751-1097.2011.00955.x

The aim of this study was to analyze the photostability and phototoxicity mechanism of anthracene (ANT) in a human skin epidermal cell line (HaCaT) at ambient environmental intensities of sunlight/UV-R (UV-A and UV-B). Photomodification of ANT under sunlight/UV-R exposure produced two photoproducts, anthrone and 9,10 anthracenedione. Generation of ¹O₂, O₂^?- and ^?OH was measured under UV-R/sunlight exposure. Involvement of reactive oxygen species (ROS) was further substantiated by their quenching with free radical quenchers. Photodegradation of 2-deoxyguanosine and linoleic acid peroxidation showed that ROS were mainly responsible for ANT phototoxicity. ANT generates significant amount of intracellular ROS in cell line. Maximum cell viability (85%) was reduced under sunlight exposure (30 min). Results of MTT assay accord NRU assay. ANT (0.01 μg mL^-1) induced cell-cycle arrest at G1 phase. RT-PCR demonstrated constitutive inducible mRNA expression of CYP 1A1 and 1B1 genes. Photosensitive ANT upregulates CYP 1A1 (2.2-folds) and 1B1 (4.1-folds) genes. Thus, the study suggests that ROS and DNA damage were mainly responsible for ANT phototoxicity. ANT exposure may be deleterious to human health at ambient environmental intensities reaching the earth's surface through sunlight. Mechanism of anthracene was assessed on HaCaT cell line at ambient environmental intensities of sunlight/UV-R. Anthracene under sunlight/UV-R exposure produced anthrone and 9,10 anthracenedione. Generation of ¹O₂, O₂^?-, and ^?OH was measured under UV-R/sunlight exposure. Photodegradation of 2-deoxyguanosine and linoleic acid peroxidation showed that ROS were mainly responsible for phototoxicity. Anthracene generates intracellular ROS in cell line. Maximum phototoxicity was observed under sunlight. Anthracene induced cell cycle arrest at G1 phase. RT-PCR study shows upregulation of CYP 1A1 and 1B1 genes. Thus, the study suggests that ROS and DNA damage were mainly responsible for anthracene phototoxicity.

Full text of DOI:10.1111/j.1751-1097.2011.00955.x

Photochemistry and Photobiology, 2011, 87: 1067–1076
Production of ROS by Photosensitized Anthracene Under Sunlight and
UV-R at Ambient Environmental Intensities
Syed Faiz Mujtaba1, Ashish Dwivedi1, Mohana Krishna Reddy Mudiam2, Daoud Ali1, Neera Yadav1
and Ratan Singh Ray*1
1Photobiology Division, CSIR – Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh, India
2Analytical Chemistry, CSIR – Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh, India
Received 18 March 2011, accepted 30 May 2011, DOI: 10.1111/j.1751-1097.2011.00955.x
products. ANT adsorbs on atmospheric particles (4), incor-
porates into soils (5), sediments (6) and produces a long-lasting
ABSTRACT
The aim of this study was to analyze the photostability and
phototoxicity mechanism of anthracene (ANT) in a human skin
epidermal cell line (HaCaT) at ambient environmental inten-
sities of sunlight ⁄ UV-R (UV-A and UV-B). Photomodification
of ANT under sunlight ⁄ UV-R exposure produced two photo-
contamination to the ecosystem, threatening a great variety of
biotopes. Concentrations of ANT from 0.9 to 75 ng m)3 have
been found in urban air (7), 3.3 lg g)1 in black tattoo inks (8)
and 0.01–19 lg kg)1 in smoked meat products (9). High
amount of PAHs are released from power stations and waste
incineration processes in the UK (10). Earlier studies of Indian
cities showed higher concentrations of PAHs in ambient
aerosol than internationally, urban cities of China having
170–490 ng m)3 and atmospheric deposition monitored near
New England coastal waters (11,12).Occupational exposure or
topical application of chemicals or drugs containing PAHs
elicit inflammatory skin diseases such as contact hypersensi-
tivity or dermatitis (13). Significant increase of skin cancer has
been reported at various industrial workplaces due to high
exposure of PAHs (14). PAHs have multiple aromatic rings
enabling them to absorb sunlight in the visible and UV regions
of solar spectrum, causing structural alterations of different
products, anthrone and 9,10 anthracenedione. Generation of
•)
1O2, O2
and •OH was measured under UV-R ⁄ sunlight
exposure. Involvement of reactive oxygen species (ROS) was
further substantiated by their quenching with free radical
quenchers. Photodegradation of 2-deoxyguanosine and linoleic
acid peroxidation showed that ROS were mainly responsible
for ANT phototoxicity. ANT generates significant amount of
intracellular ROS in cell line. Maximum cell viability (85%)
was reduced under sunlight exposure (30 min). Results of MTT
assay accord NRU assay. ANT (0.01 lg mL)1) induced cell-
cycle arrest at G1 phase. RT-PCR demonstrated constitutive
inducible mRNA expression of CYP 1A1 and 1B1 genes.
Photosensitive ANT upregulates CYP 1A1 (2.2-folds) and 1B1
(4.1-folds) genes. Thus, the study suggests that ROS and DNA
damage were mainly responsible for ANT phototoxicity. ANT
exposure may be deleterious to human health at ambient
environmental intensities reaching the earth’s surface through
sunlight.
•)
biomolecules. Solar UV-R increased production of O2 and
lipid peroxidation in ANT treated fish microsomes (15).
Although the role of PAHs as photosensitizers has received
little attention, there is a report that excitation of PAHs by
sunlight produced phototoxicity and DNA damage (16). The
PAHs under solar radiation (a light source with a spectrum
similar to sunlight) inhibited the leaf production of duckweed
(Lemna gibba) (17). PAHs undergo various photochemical
reactions under UV-R exposure involving the generation of
reactive oxygen species (ROS) and photoproducts which can
produce membrane damage, DNA damage and cell death.
UV-A photoirradiation of oxygenated benz(a)anthracene and
INTRODUCTION
Human activities, resulting in the release of chlorofluorocar-
bons and nitrogen oxides into the stratosphere, have signifi-
cantly decreased the ozone concentration in the past 30 years
(1). UV-C and UV-B are absorbed largely within the epider-
mis, whereas 50% of UV-A penetrates into the dermis. UV-B
radiation at the earth’s surface has increased 6–14% since 1980
due to ozone depletion (2). At present, ozone-depleting
substances have been reduced 95% by the Montreal Protocol.
However, many ozone-depleting substances already in the
atmosphere are long-lived, and present projections estimate a
return to pre-1980 levels by 2050–2075 (3). Anthracene (ANT)
is a polycyclic aromatic hydrocarbon (PAH), produced by
incomplete burning of fossil fuels and discharge of petroleum
1
3-methylcholanthin generates O2 and induced lipid peroxida-
tion (18). PAHs may be metabolized by cytochrome P450
enzymes into reactive intermediates such as diol-epoxides and
diones that form covalent DNA-adducts, which may lead to
mutagenesis and carcinogenesis (19). Toxicological studies
defining the potential hazards of PAHs have been conducted
in the absence of UV radiation. Therefore, comparative study
of PAHs’ effects both in the presence and absence of
UV-R ⁄ sunlight is important.
Recently, some PAHs listed by the EPA as priority
pollutants were reported to be photomutagenic (20). The
activity and expression of CYP1A1, 1A2 and 1B1, are induced
by PAHs (21). The photosensitized tryptophan photoproducts
*Corresponding author email: ratanray.2011@rediffmail.com (Ratan S. Ray)
ꢀ 2011 The Authors
Photochemistry and Photobiology ꢀ 2011 The American Society of Photobiology 0031-8655/11
1067

1068 Syed F. Mujtaba et al.
Photochemical assay. Determination of singlet oxygen (1O2): The
generation of 1O2 under aerobic condition was measured in aqueous
solution by the method of Ray et al. (23). RNO solution (0.35–
0.4 · 10)5 M) was prepared in 0.025 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
compound was irradiated under UV-A (7.92 J cm)2), UV-B
(3.24 J cm)2) and sunlight (60 min). The production of 1O2 was
monitored by measuring the decrease in RNO absorbance at 440 nm
by spectrophotometer (Varian UV-visible- carry-300 BIO). The
generation of 1O2 was further substantiated by the administration of
DABCO (25 and 50 mM) as a specific quencher.
modulate light-dependent regulation of circadian rhythm
through triggering of AhR signaling (22).
This study investigated the phototoxic mechanism of ANT
on human keratinocyte (HaCaT) cell line, focusing on the
involvement of UV-R ⁄ sunlight induced oxidative stress-
related phototoxicity, cell-cycle arrest and mRNA expression
of CYPs genes.
MATERIALS AND METHODS
Determination of superoxide anion radical (O2•)): The generation of
Chemicals and culture wares. ANT, N,N-dimethyl-p-nitrosoaniline
(RNO), superoxide dismutase (SOD), nitro-blue tetrazolium (NBT),
fetal bovine serum (FBS), Dulbecco’s modified eagle’s medium
(DMEM F-12 HAM), antibiotic and antimycotic solution, trypsin
(0.25%), L-histidine, 3-(4,5-dimethylthiozolyl-2)-2,5-diphenyl-2H-tet-
razolium bromide (MTT), neutral red uptake (NRU), 1,4-diazabicyclo
2-2-2-octane (DABCO), mannitol, sodium benzoate, tricarboxylic acid
(TCA), ascorbic acid, carbonate and phosphate buffers, RNase,
propidium iodide, 5(and-6)-carboxy,2 7-dichlorodihydrofluorescein
diacetate (carboxy-H2 DCFDA), N-acetyl-cysteine (NAC) and
2¢-deoxyguanosine (2¢-dGuO) were procured from Sigma Chemical
Co. (St. Louis, MO). Linoleic acid and Tween-20 were obtained from
M.P. Biomedicals Inc. (Solon) and Hank’s Balanced Salt Solution
(HBSS), PBS were purchased from In Vitrogen Corporation. Other
reagents and chemicals were procured from Hi-Media, Ranbaxy and
Merck India Ltd. All plastic wares including well plates and culture
flasks (polystyrene coated) were purchased from Nunc.
•)
O2 was monitored by recording the photosensitized reduction of
NBT to nitroblue diformazan (NBF) spectrophotometrically. NBT
solution (1.67 · 10)4 M) was prepared in 0.01 M sodium carbonate
buffer (pH = 10). A 10 mL assay system containing ANT from 0.1 to
1.0 lg mL)1 was irradiated under UV-A (2.64 J cm)2), UV-B
(1.08 J cm)2) and sunlight (20 min). The production of NBF was
monitored by measuring the increase in absorbance at 560 nm. The
·)
generation of O2 was further confirmed by quenching with SOD (10
and 25 Units mL)1) (24).
•
Determination of hydroxyl radical (•OH): The OH generation was
measured by ascorbic acid-iron-EDTA system. The iron-catalyzed
oxidation of ascorbic acid was used at 37ꢁC. The standard reaction
mixture contains 100 mM potassium phosphate buffer pH 7.4, 167 lM
iron-EDTA (1:2), 0.1 mM EDTA, 2 mM ascorbic acid and 33 mM
dimethyl sulfoxide in a final volume of 3.0 mL and irradiated. After
irradiation, 1.0 mL of TCA (17.5%, wt ⁄ vol) was added. The samples
were then assayed for formaldehyde formation. Ammonium acetate
acetylacetone reagent was prepared by 2.0 M ammonium acetate,
0.05 M acetic acid and 0.02 M analytical grade redistilled acetyl
acetone. Equal volumes of sample (1.5 mL) and reagent (1.5 mL)
were mixed and kept at 37ꢁC for 40 min. The production of
formaldehyde was monitored at 412 nm. The quenching of •OH was
performed by addition of mannitol (0.5 M) and sodium benzoate
(0.5 M) as specific quenchers (25).
Photodegradation of 2¢-dGuO: The photo-oxidative degradation of
2¢-dGuO by ANT was studied. A 10 mL solution of 2¢-dGuO in 0.01 M
carbonate buffer (pH 10.0) with or without ANT, was placed in petri
dishes. The samples were irradiated under UV-A (7.92–31.68 J cm)2),
UV-B (3.24–12.96 J cm)2) or sunlight (60–240 min) for different time
intervals. The degradation of 2¢-dGuO was monitored spectrophoto-
metrically at 260 nm. Photodegradation quenching of 2¢-dGuO was
substantiated by DABCO (25 and 50 mM) (25).
Linoleic acid photoperoxidation. Linoleic acid solution was freshly
prepared in phosphate buffered saline (0.01 M, pH 7.2) using 0.05%
tween-20 as an emulsifying agent. A solution containing 0.8 mM
linoleic acid with ANT was irradiated under UV-A (3.96–
15.84 J cm)2), UV-B (1.62–6.48 J cm)2) and sunlight (30–60 min).
Light-induced photoperoxidation of linoleic acid was measured by
the increase in absorbance at 233 nm (26). Quenching of linoleic
acid photoperoxidation was performed using SOD (25 and
50 Units mL)1).
Determination of ROS production in cells. Cells were grown in six
multiwell plates (6 · 105 cells per well) and treated with ANT (0.01–
0.5 lg mL)1). Cells were then incubated for 30 min at 37ꢁC with
5 lM carboxy H2-DCFDA in HBSS and exposed under sunlight,
UV-A and UV-B. After irradiation of cells, the fluorescence was
measured by fluorometer (Fluostar Omega-BMG Labtech) using at
excitation and emission wavelengths of 480 nm and 530 nm, respec-
tively (27).
Cell culture. The HaCaT cell line was procured from National
Centre for Cell Sciences Pune, India (passage number 46). It was sub
cultured and maintained in our laboratory. The cell line was grown in
DMEM culture medium supplemented with 10% FBS and antibiotic–
antimycotic solution (1.5%) at 5% CO2 and 95% relative humidity
(37ꢁC).
Source and radiation exposure. The UV-R 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.
Spectral emissions ranged from 320 to 400 nm with a peak at 365 nm
for the UV-A source and from 290 to 320 nm with a peak at 312 nm
for the UV-B source. Intensities selected for irradiations were based on
dosimetry carried out at our laboratory’s roof top between 12.00 noon
to 1.00 pm (5 days a week) and were parallel to the ambient intensities
of UV-A and UV-B in sunlight reaching at Lucknow (26o45¢N latitude
and 80o50¢E longitude at 146 m above the mean sea level).
Radiation exposure was carried out in a temperature controlled
radiation chamber at 25ꢁC
2ꢁC. The samples were put at a
minimum distance of 22.0 cm from the source. Glass petri dishes
(60 · 15 mm) were used for photochemical reactions. The cells were
exposed in 96 well plates. Sunlight exposure was carried out during
clear sunny days between 11.00 A.M to 3.00 P.M. Petri dishes ⁄ culture
plates were put on a platform surrounded by ice packs (Polar tech
Industries, Genova IL, Los Angeles) to prevent thermal effect.
Analysis of ANT through GC-MS. ANT (5 lg mL)1) was dissolved
in DMSO and exposed to sunlight, UV-A & UV-B. Aliquots of
irradiated ANT were drawn at the intervals of 60 min from 0 to
240 min. GC-MS analysis was performed by Perkin Elmer Auto XL
system (Gas chromatography) coupled with Turbo Mass (Mass
spectrophotometer). Chromatographic resolution achieved by PE-5
MS (5% phenylmethyl siloxane) capillary column (30 · 250 lM) with a
film thickness of 0.32 lM. Helium was used as a carrier gas with flow
rate 1.1 mL min)1. The column temperature was kept at 70ꢁC for
1 min and then programmed to 200ꢁC at a rate of 25ꢁC min)1 and
260o at a rate of 8ꢁC min)1. Total run time of the analysis was
26.7 min with a solvent delay of 5 min. The spectrum was recorded
Phototoxicity assay. Cultured cells were seeded in 25 cm2 tissue
culture flasks. The cells were trypsinized by trypsin-EDTA solution
(0.25%), after 48 h at 80–90% confluency, mixed with equal volume of
DMEM and F-12 HAM’s medium and collected in 50 mL sterilized
plastic tubes. Cells were washed twice with HBSS. 2 · 104 cells were
seeded per well in 96 well plates. After incubation for 48 h, the medium
was aspirated and cells were washed with HBSS, prior to starting the
experiment. Controls included a basal control (cells only), dark control
(cells treated with ANT), light control (cells exposed to light only),
solvent control (cells treated with solvent only) and solvent light
control (cells exposed to light with solvent) along with the experimen-
tal sets. Prior to UV-R, treated cells in HBSS were incubated for
with
a mass range of 50–400 (m ⁄ z) in scan mode. The mass
spectrometer was operated in electron ionization mode with electron
energy 70 eV. Injector temperature was set at 260ꢁC. The transfer line
and ion source temperatures were maintained at 200ꢁC and 250ꢁC,
respectively. A solvent delay of 3 min was selected. Identification of
photoproducts was performed by comparing their mass spectra with
the NIST library available in the instrument.

Photochemistry and Photobiology, 2011, 87 1069
30 min in CO2 incubator to permit the cells to absorb ANT. Cells were
then exposed to sunlight ⁄ UV-R for the desired time followed by
incubation of 30 min in a CO2 incubator. HBSS was replaced by
DMEM F-12 HAM and batches were processed for phototoxicity
assays.
which was confirmed through its NIST library, similarly the
mass spectrum of 9,10 anthracenedione (b) and anthrone (c)
were also confirmed through NIST library. ANT (m ⁄ z = 178)
photodegraded to anthrone (m ⁄ z = 194) and 9,10 anthracen-
edione (m ⁄ z = 208) within 90 min of irradiation. Further
exposure of ANT up to 4 h led to total conversion of ANT and
anthrone to 9,10 anthracenedione under sunlight exposure
(Fig. 1D).
MTT assay. In brief, cells (2 · 104) were seeded per well in 96 well
plates and kept in the CO2 incubator for 48 h at 37ꢁC for 80–90%
confluency. The medium was replaced by HBSS with ANT for
exposure. At the end of irradiation, HBSS was replaced by 20 lL MTT
(5 mg mL)1) in 200 lL complete medium. The culture plates were kept
in the CO2 incubator for 4 h. After incubation, the culture plates were
washed twice with HBSS and 200 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 (Fluostar
Omega- BMG Labtech) (25).
Figure 2a shows the mean photochemical generation of 1O2
by ANT at different concentrations (0.1–1 lg mL)1) under
sunlight (60 min) and UV-A (7.92 J cm)2) exposures. ANT at
1 lg mL)1 generates the highest amount of 1O2 under sunlight
exposure followed by UV-A. Further the generation of 1O2
increases significantly (P < 0.05 or P < 0.01) with concen-
NRU assay. Briefly, after irradiation, the cells containing ANT were
washed with HBSS. The culture well plates were allowed to incubate
for 3 h in complete medium (DMEM F-12 HAM) containing neutral
red dye (50 lg mL)1) followed by a quick wash with fixative (1%
wt ⁄ vol CaCl2; 0.5% vol ⁄ vol formaldehyde) to remove the unbounded
dye. The dye accumulated in cells 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 (25).
1
trations. Highest O2 generation was recorded under sunlight
exposure (b = 0.2155) followed by UV-A (b = 0.1818). No
significant 1O2 generation was observed under UV-B exposure.
In an attempt to evaluate the possible role of ROS in ANT
phototoxicity, specific 1O2 quencher was used (Fig. 2b).
Cell-cycle analysis. ANT (0.01 and 0.05 lg mL)1) treated cells were
kept in the dark and then exposed to UV-A (3.96 J cm)2). After UV
exposure the cells were washed twice with PBS, and replaced with fresh
culture medium. After incubation of 18 h, cells were harvested with
trypsin and fixed in 70% ethanol. The fixed cells were then washed
twice with PBS and treated with RNase (0.1 mg mL)1) for 30 min at
37ꢁC. Finally, the cells were stained with propidium iodide
(50 lg mL)1) and incubated in the dark for 60 min prior to analysis.
Cellular DNA content was determined by flow cytometry (Becton–
Dickinson LSR-II) using the Cell Quest program and Mod Fit
software.
RNA Isolation and cDNA synthesis. Total RNA was isolated by
using phenol–chloroform extraction. Purity and concentration of the
isolated RNA was determined by nano-drop spectrophotometer (ND-
1000 Thermo scientific) at 260 nm. RNA samples were stored at
)20ꢁC. For real-time PCR, cDNA was synthesized by high-capacity
cDNA reverse transcription kit (28).
Quantitiative real-time PCR analysis. Fold induction was measured
for CYP-1A1 and CYP 1B1 genes by real-time PCR (Applied
Biosystems—7900 HT Fast-Real-time PCR system) using ABI—
sequence detection system (PE Applied Biosystems, Foster City,
CA). Real-time PCR consists of initial denaturing for 10 min at 95ꢁC,
40 cycles of 95ꢁC for 15 s and 50ꢁC for 1 min. Each sample was
assayed in triplicates. The cycle threshold (CT) values were normalized
to the housekeeping gene (GAPDH) and the fold change was
calculated using 21DDcT method (29).
1
Results showed 48% quenching of O2 by DABCO (50 mM)
with 1 lg mL)1 ANT under UV-A (7.92 J cm)2) exposure.
Generation of O2 is summarized in Fig. 3a. Results
•)
•)
showed that mean generation of O2 by ANT was compar-
atively higher in sunlight (60 min) followed by UV-A
(2.6 J cm)2) and UV-B (1.08 J cm)2). The order of O2
•)
generation was the following: sunlight > UV-A > UV-B.
•)
Further, the fold generation of O2 increased significantly
(P < 0.05 or P < 0.01) with ANT concentrations, and was
found highest in UV-A (b = 0.297) followed by UV-B
•)
(b = 0.196) and sunlight (b = 0.1758). The increase of O2
in UV-A was 1.52 and 1.69 times higher than UV-B and
sunlight, respectively. Additional evidence of the production of
•)
•)
O2 was obtained by examining the O2 quenching study
with SOD (10 and 25 Units mL)1) under UV-A (2.46 J cm)2
)
was measured by
•)
irradiation. 50% quenching of O2
25 Units mL)1 of SOD (Fig. 3b).
Generation of •OH is summarized in Fig. 4a. Results
showed that mean OH generation was comparatively higher
•
in sunlight (60 min) followed by UV-A (7.92 J cm)2) and
UV-B (3.24 J cm)2). Further the fold generation in all three
exposures increased significantly (P < 0.05 or P < 0.01)
with concentrations, and was found highest in sunlight
(b = 121.86) followed by UV-A (b = 117.05) and UV-B
Statistical analysis. Group means
SE were compared by one-
way analysis of variance (ANOVA) followed by Tukey’s post hoc
test. Correlation and simple linear regression analysis were used to
assess the cell viability, ROS generation, 2¢-dGuO photodegradation
and lipid peroxidation against different ANT concentrations. A two-
tailed (a) probability P < 0.05 was considered statistically signifi-
cant.
•
(b = 83.055). The increase of OH generation in sunlight was
1.041- and 1.467-folds higher than UV-A and UV-B, respec-
•
tively. Figure 4b shows the percent quenching of OH under
UV-A (7.92 J cm)2) at 1 lg mL)1 of ANT by mannitol (0.5 M)
and sodium benzoate (0.5 M) caused 28% and 42% quenching,
respectively. Higher quenching was observed by sodium
benzoate than mannitol.
RESULTS
ANT showed strong absorption maxima (kmax) in UV-A
(360 nm) (Fig 1A). ANT (5 lg mL)1) was exposed to sun-
light ⁄ UV-R for various time period from 0 to 240 min.
GC-chromatogram of ANT showed the results at 60 min
interval. ANT (control) without irradiation (Fig. 1B(a)) had a
retention time of 8.90 min. After 120 min of irradiation the
two photoproducts were detected with retention times of 10.30
(9,10 anthracenedione) and 8.52 (anthrone) (Fig. 1B(c)). Total
conversion of ANT to anthracenedione was observed at
240 min. ANT and anthrone were photodegraded to 9,10
anthracenedione after 240 min exposure with retention time
10.32. Figure 1B(e),C(a) shows the mass spectra of ANT
Photodynamic degradation of 2¢-dGuO by ANT
(1 lg mL)1) is summarized in Fig. 5a. Results showed that
maximum degradation of 2¢-dGuO was recorded under sunlight
(60–240 min) followed by UV-A (7.92–31.68 J cm)2) and
UV-B (3.24–12.96 J cm)2). Further the fold degradation in all
three exposures increased significantly (P < 0.05 or P < 0.01)
with time period and was found highest in UV-A (b = 0.4041)
followed by UV-B (b = 0.2107) and sunlight (b = 0.1885).
The increase of 2¢-dGuO degradation in UV-A was 1.9178-
and 2.1437-folds higher than UV-B and sunlight, respectively.
Fig. 5b showed the percent quenching of photodynamic

1070 Syed F. Mujtaba et al.
(A)
(C)
(a) anthracene
(b) 9,10 anthracenedione
(B)
(e)
(d)
(c) anthrone
Mass Spectra
(c)
(b)
(D)
(a)
m/z=208
m/z=178
ANT
m/z=194
Anthrone
9,10 Anthracenedione
m/z=208
9,10 Anthracenedione
Figure 1. (A) Absorption spectrum of ANT. (B) GC values of ANT photoproducts irradiated under sunlight exposure. (a) control (b) 60 min (c)
120 min (d) 180 min (e) 240 min. (C) Mass spectra of ANT and its photoproducts compared with NIST library. (D) Various photoproducts of
ANT radiated under sunlight for 0–240 min.
degradation of 2¢-dGuO by DABCO (25 and 50 mM) under
UV-A exposure. Maximum 60% quenching of 2¢-dGuO
photodegradation was observed by DABCO (50 mM).
and UV-B, respectively. Linoleic acid peroxidation quenching
was carried out under UV-A (7.92 J cm)2) by SOD (25 and
50 Units mL)1). Significant quenching was observed at
50 Units mL)1 of SOD (Fig. 6b).
Figure 6a shows the linoleic acid photoperoxidation by
ANT (1 lg mL)1) under sunlight (30–120 min), UV-A (3.96–
Figure 7a shows the DCF-fluorescence intensity at different
concentrations of ANT under sunlight (60 min), UV-A
(7.92 J cm)2) and UV-B (3.24 J cm)2) exposures. As com-
pared with control, a significant (P < 0.05 or P < 0.01)
increase of DCF intensity was observed in all treated cells in a
concentration dependent manner. Maximum DCF intensity
was recorded under sunlight at 0.5 lg mL)1 of ANT followed
by UV-A and UV-B irradiations. Results indicate that ANT
facilitates UVR-induced oxidative stress in HaCaT cell line. In
the presence of an antioxidant, NAC inhibited DCF-fluores-
cence intensity in a dose-dependent manner under sunlight
(60 min) exposure (Fig. 7b).
15.84 J cm)2
)
and UV-B (1.62–6.48 J cm)2
)
exposures.
Formation of dienic hydroperoxide was maximum under
sunlight exposure. Linoleic acid itself photoperoxidized under
UV-R ⁄ sunlight but higher yield was observed with ANT. The
photoperoxidation of linoleic acid was in the following order:
sunlight > UV-A and UV-B. Further the fold peroxidation in
all three exposures increases significantly (P < 0.05 or
P < 0.01) with increasing time period, and was found highest
in sunlight (b = 0.008) followed by UV-A (b = 0.0058) and
UV-B (b = 0.0038). The increase of linoleic acid peroxidation
in sunlight was 1.3793- and 2.1052-folds higher than UV-A

Photochemistry and Photobiology, 2011, 87 1071
160
140
120
100
80
0.60
0.50
0.40
0.30
0.20
0.10
0.00
(a)
0.1 μg/ml
0.1 μg/ml
0.5 μg/ml
1 μg/ml
**
**
(a)
0.5 μg/ml
**
1 μg/ml
*
*
*
**
60
**
*
40
*
20
0
Sunlight
UV-A
UV-B
Sunlight
UV-A
Radiation exposure
Radiation exposure
60.00%
(b)
**
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
(b)
50
45
40
35
30
25
20
15
10
5
**
25
50
0
DABCO (mM)
Figure 2. (a) Photochemical generation of 1O2 by ANT at different
Mannitol
Sodium benzoate
concentrations under sunlight (60 min) and UV-A (7.92 J cm)2
)
Quenchers concentration (0.5 M)
exposure (*P < 0.05 or **P < 0.01 as compared to 0.1 lg mL)1).
(b). Percent photochemical quenching of 1O2 generated by ANT
(1 lg mL)1) through DABCO under UV-A (7.92 J cm)2) exposure
(**P < 0.01 as compared to 25 mM DABCO). Each value presented is
•
Figure 4. (a) Photochemical generation of OH at various concentra-
tions of ANT under sunlight (60 min), UV-A (7.92 J cm)2) and UV-B
(3.24 J cm)2
)
(*P < 0.05 or **P < 0.01 as compared to
0.1 lg mL)1). (b). Percent quenching of ·OH at 1 lg mL)1 of ANT
by mannitol and sodium benzoate under UV-A (7.92 J cm)2
(**P < 0.01 as compared to mannitol). Each value presented is mean
of three observations SE.
mean of three observations
SE.
)
0.6
0.5
0.4
0.3
0.2
0.1
0
(a)
0.1 μg/ml
0.5 μg/ml
1 μg/ml
**
*
percent cell viability. The phototoxicity was assessed under
sunlight (30 min), UV-A (3.96 J cm)2) and UV-B (1.62 J cm)2
exposures. Cytotoxicity was also assessed with different
ANT concentrations without irradiation. Chlorpromazine
(1 lg mL)1
**
)
*
**
) ) were used as
and L-histidine (10 lg mL)1
*
positive and negative photosensitizers, respectively. The high-
est decrease (P < 0.01) in percent cell viability by ANT
(0.5 lg mL)1) was observed under sunlight (85%) followed by
UV-A (60%) and UV-B (55%) exposures. It indicates that
UV-B irradiated ANT (0.5 lg mL)1) was least phototoxic,
while ANT (0.1 lg mL)1) under sunlight was highly photo-
toxic. Phototoxicity of ANT was observed in the following
order: Sunlight > UV-A > UV-B. In all three exposures as
concentration increases % cell viability reduced significantly
(P < 0.05 or P < 0.01) than negative control (L-histidine).
The regression b coefficient which indicates the rate of change
also showed highest reduction in cell viability under sunlight
(b = )19.56) followed by UV-A (b = )15.41) and UV-B
(b = )11.84) were found 35.14-, 27.68- and 21.27-fold higher,
respectively as compared to dark (b = )0.55).
Figure 8b shows the relative photosensitizing effect of ANT
at various concentrations (0.01–0.5 lg mL)1) on HaCaT cell
line by recording NRU as percent cell viability under sunlight
(30 min), UV-A (3.96 J cm)2) and UV-B (1.62 J cm)2). It is
evident from data that ANT under sunlight exposure was
highly phototoxic followed by UV-A and UV-B (sun-
light > UV-A > UV-B). In all three exposures as concentra-
tion increases % cell viability reduced significantly (P < 0.05
or P < 0.01) when compared to negative control (L-histidine).
The regression b coefficient which indicates the rate of change
Sunlight
UV-A
UV-B
Radiation exposure
60
50
40
30
20
10
0
(b)
**
10
25
SOD (Units/ml)
•)
Figure 3. (a) Photochemical generation of O2 by ANT at various
concentrations under sunlight (20 min), UV-A (2.6 J cm)2) and UV-B
(1.08 J cm)2
)
(*P < 0.05 or **P < 0.01 as compared to
0.1 lg mL)1). (b) Photochemical quenching of O2 generated by
ANT (1 lg mL)1 through SOD under UV-A (2.6 J cm)2
(**P < 0.01 as compared to 10 Units mL)). Each value presented is
mean of three observations SE.
•)
)
)
Figure 8a shows the ANT phototoxicity on HaCaT cell
line through MTT assay. Photosensitizing effect of ANT at
various concentrations (0.01–0.5 lg mL)1) was recorded as

1072 Syed F. Mujtaba et al.
1200
1000
800
600
400
200
0
100
(a)
90
**
**
60 min
(a)
*
HBSS
0.01
0.05
0.1
**
120 min
180 min
240 min
**
**
**
80
70
60
50
40
30
20
10
0
**
**
**
**
**
0.5
**
*
**
**
*
*
*
Sunlight
UV-A
UV-B
Sunlight
(b)
UV-A
UV-B
Radiation exposure
Radiation exposure
(b)
1200
70.00%
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
**
1000
800
600
400
200
0
*
**
**
25
50
DABCO (mM)
0
1
10
100
Figure 5. (a) Photodynamic degradation of 2¢dGuO by photoillu-
minated ANT (1 lg mL)1
under sunlight, UV-A and UV-B
)
NAC (µM)
exposure (*P < 0.05 or **P < 0.01 as compared to 60 min). (b)
Percent quenching of photodynamic degradation 2¢dGuO by DABCO
under UV-A (7.92 J cm)2) exposure (**P < 0.01 as compared to
25 mM DABCO). Each value presented is mean of three observa-
Figure 7. (a) DCF-Fluorescence intensity at different concentrations
of ANT (lg mL)1) under sunlight (60 min), UV-A (7.92 J cm)2) and
UV-B (3.24 J cm)2) exposure (*P < 0.05 or **P < 0.01 as compared
to HBSS). (b). Intracellular quenching of DCF fluorescence by NAC
under sunlight (60 min) exposure at 0.5 lg mL)1 ANT (*P < 0.05 or
**P < 0.01 as compared to 0 lM). Each value presented is mean of
tions
SE.
three observations
SE.
1.4
(a)
30 min
60 min
120 min
also showed that highest reduction in cell viability under
sunlight (b = )20.29) followed by UV-A (b = )19.75) and
UV-B (b = )12.05) were found 34.21-, 33.28- and 20.31-folds
higher, respectively as compared to dark (b = )0.59).
Figure 9 a,b,c & d show the cell-cycle study through flow
cytometry by propidium iodide staining. Cells were exposed to
UV-A (3.96 J cm)2) irradiation and ANT (0.01 lg mL)1 and
0.05 lg mL)1). The number of cells at the G1 phase increased
significantly than dark (control) cells. This increase occurred
concomitantly with decrease in number of cells in S and G2
phases. ANT (0.05 lg mL)1) under similar dose showed a
striking decrease in the number of cells in G1, S and G2 phases
in comparison to dark which confirmed the cell death due to
ANT phototoxicity. A slight increase of cells in Go phase
indicates the induction of apoptosis.
Figure 10 shows the photosensitized ANT induced genes
expression of CYP 1A1 and 1B1 in HaCaT cell line under
UV-A (3.96 J cm)2) exposure through real-time PCR. UV-A
and ANT (0.01 lg mL)1) treated cells were kept for 6 h
incubation showed upregulation of CYP 1A1 (2.2-fold) and
CYP 1B1 (4.1-fold) genes, while concomitantly in dark the
expression was significantly lower in both CYP 1A1 (1.0-fold)
and CYP 1B1 (1.5-fold) genes. Each sample was normalized
with housekeeping gene GAPDH, which served as an endog-
enous control. Expression of GAPDH was found uniform in
all the samples analyzed, confirming the integrity of mRNA
used in assays.
**
1.2
1
**
**
0.8
0.6
0.4
0.2
0
**
*
Sunlight
UV-A
UV-B
**
Radiation exposure
35
30
25
20
15
10
5
(b)
0
25
50
SOD (Units/ml)
Figure 6. (a) Photoperoxidation of linoleic acid by ANT (1 lg mL)1
under sunlight, UV-A and UV-B exposure. The formation of dienic
hydroperoxides was followed by the increase in absorption at 233 nm.
(*P < 0.05 or **P < 0.01 as compared to 30 min). (b) Photochemical
quenching of linoleic acid (0.8 mM) peroxidation by SOD under UV-A
(7.92 J cm)2) (**P < 0.01 as compared to 25 Units mL)1 of SOD).
)
Each value presented is mean of three observations
SE.

Photochemistry and Photobiology, 2011, 87 1073
120.00
100.00
80.00
60.00
40.00
20.00
0.00
(a)
Chlorpromazine
L-hisꢀdine
0.01
0.05
0.1
p>0.05
*
*
*
**
**
0.5
**
**
**
**
**
**
**
Dark
Sunlight
UV-A
UV-B
Radiation exposure
Chlorpromazine
L-Hisꢀdine
0.01
0.05
0.1
120
100
80
60
40
20
0
(b)
p>0.05
*
*
**
**
**
**
0.5
**
**
**
**
Dark
Sunlight
UV-A
UV-B
Radiation exposure
Figure 8. Effect of photosensitized ANT on keratinocyte viability through MTT (a) and NRU (b) assay under dark, sunlight (30 min), UV-A
(3.96 J cm)2) and UV-B (1.62 J cm)2) exposures. Chlorpromazine (1 lg mL)1) and L-histidine (10 lg mL)1) were used as positive and negative
controls, respectively. Each value presented are mean of three observations
SE. (*P < 0.05 or **P < 0.01 as compared to L-histidine).
produced O2 and •OH (Type I photodynamic reaction).
•)
DISCUSSION
DABCO was able to control the generation of 1O2 while
mannitol and sodium benzoate were able to quench •OH.
Quenching studies with specific quenchers of 1O2 and •OH
showed that 1O2 and •OH were predominant in ANT
Continuous depletion of stratospheric O3 layer has increased
the ambient levels of UV-R on earth’s surface. Depletion of
1% O3 layer increases 3% nonmelanoma skin cancers (30),
and 0.25–0.6% cataracts. We have demonstrated the ANT
phototoxicity and its mechanism under ambient environmental
intensities of UV-A, UV-B encountered in Lucknow’s sunlight.
Earlier studies of phototoxicity evaluation were performed at
higher UV-R doses (31,32). The aim of the present study was
to gain more insight into the process of photodegradation,
identification of its photoproducts and role of ROS in the
manifestation of ANT phototoxicity. Sunlight is a natural
source of different wavelengths containing UV-A,UV-B and
many other radiations, therefore the effect of sunlight with
photosensitizer may be cumulative and higher. Our study
indicates that ANT was generating 1O2, O2•) and •OH through
photosensitized mechanisms, in which the photoexcited ANT
(triplet state) transfers its energy to O2 and generates energy
•
phototoxicity. The high yield of 1O2 and OH indicates that
phototoxic reactions occurring in the cells might be mediated
by ROS. 2¢dGuO was used to assess nuclear damage due to the
relative high sensitivity of guanine base to sunlight ⁄ UV-R. The
significant photodegradation of 2¢dGuO was observed by
photosensitized ANT. Furthermore, quenching by specific 1O2
quencher (DABCO) confirms the involvement of 1O2 in the
photodegradation of 2¢dGuO. 1O2 is known to damage cellular
structure by oxidation of lipids and proteins, and may be
1
responsible for many deleterious effects in tissues (33). O2 is
an important ROS in DNA damage under in vitro conditions
(34). The photoirradiated PAHs by UV-A light generate ROS
which induced lipid peroxidation (35). The phototoxicity of
ANT on cell line and lipid peroxidation of linoleic acid
suggests that the ROS might initiate cellular lipid peroxidation
which finally leads to cell death. To examine the validity of this
hypothesis, linoleic acid photoperoxidation was used as a
model system (26). The ability of photosensitized ANT to
1
rich O2 (Type II photodynamic reaction), an active oxidizing
agent. Another oxygen-dependent reaction involves the pho-
toreaction of an electronically active photosensitizer, reduced
either by an electron or hydrogen transfer from a compound,

1074 Syed F. Mujtaba et al.
Figure 9. (a, b, c & d) Cell-cycle study through flow cytometry by propidium iodide staining in HaCaT cell line (18 h incubation).
significant amounts of 1O2 and O2•) and caused DNA damage
(37). These reactive forms of oxygen may be responsible for
linoleic acid peroxidation (26). Tattooing with black ink
2.5
**
2
1
generates deleterious O2 inside the dermis when skin exposed
1.5
**
to solar UV-A radiation (8). The DCF fluorescence in the cells
was increased in a concentration dependent manner which
indicates the ROS generation by photosensitive ANT. It also
indicates free radical induced oxidative stress in the cell. The
1
0.5
*
0
Dark
Dark + Ant
UV-A
UV-A + Ant
UV-R ⁄ sunlight increased dose-dependent ROS generation in
ANT treated cells. The quenching with a specific antioxidant
NAC reduced the fluorescence intensity of the DCF, which
suggests the ROS induced phototoxicity of ANT. ROS
production has been associated to PAHs and UV-R coexpo-
sure induced cell death (38). ANT was significantly reducing
the cell viability of HaCaT cell line. The human monocytic cell
line used for in vitro evaluation of the photoallergic potential
of chemicals (39).
Exposure
4.5
4
**
(b)
3.5
3
2.5
2
**
1.5
1
*
0.5
0
FACS analysis demonstrates that photosensitized ANT
(0.01 lg mL)1) caused arrest of the cell cycle in G0 ⁄ G1 phases
with decrease of cell population in synthesis and mitosis
phases. Higher ANT concentration (0.05 lg mL)1) resulted in
cytotoxicity, which showed decreased cell number in G1, S and
G2 phases, whereas control cells were unaffected. Although
HaCaT cell line is a p53 mutant, however, a transient G1 to
S-phase delay was observed after exposure of cells with
photosensitized ANT. These results indicate that DNA dam-
aging agents can inhibit G1 to S-phase progression through
p53 independent mechanism (40).
Photosensitized ANT induced higher expression of CYP 1B1
as compared to CYP 1A1. Earlier studies at ambient level of
UV-R with ANT did not report mRNA expression of both the
genes. It has been reported that CYP 1A1and 1B1 genes can
induced via AhR in A549 cells (41). CYP 1A1 and 1B1 are
expressed by PAHs in freshly prepared rat blood lymphocytes
(21). It has been hypothesized that the expression levels of AhR
Dark
Dark + ANT
UV-A
UV-A + ANT
Exposure
Figure 10. (a, b) Photosensitized ANT (0.01 lg mL)1) induced genes
expression analysis of CYP 1A1 (a) and CYP 1B1 (b) in HaCaT cell
line under UV-A (3.96 J cm)2) exposure through Real-time PCR.
Each value presented are mean of three observations
(*P < 0.05 or **P < 0.01 as compared to dark).
SE.
induce photodynamic lipid peroxidation and cell damage may
be expected in vivo. Percent quenching of linoleic acid
peroxidation by SOD accorded the involvement of ROS.
Previous study supports our viewpoint that photosensitive
diclofenac caused DNA fragmentation, leading to cellular
damage via the generation of ROS in vivo (36). Commonly
used antibiotics generate significant amount of 1O2 and caused
photodegradation of 2¢dGuO under UV-R exposure (23).
Hematoporphyrin,
a
known photosensitizer, produced

Photochemistry and Photobiology, 2011, 87 1075
European priority polycyclic aromatic hydrocarbons in smoked
meat products and edible oils. Food Addit. Contam. Part A Chem.
Anal. Control Expo. Risk Assess 25, 704–713.
and Arnt contribute to the differences in CYP 1A1 and 1B1
inducibility in human lymphocytes (42). PAHs metabolism
catalyzed by CYP 450 (CYP1A1) enzymes, generate genotoxic
metabolites and ROS (43). The exposure of window sunlight
photooxidized tryptophan photoproducts leads to induction of
CYP1A in primary chick embryo hepatocyte and in vivo (44). A
high reduction of cell viability under sunlight exposure has
demonstrated that sunlight was more lethal than UV-A and UV-
B. The selection of different intensities of UV-R prove our
viewpoint that higher intensities of UV-R in sunlight (in view of
ozone depletion) would be more deleterious.
10. Dyke, P. H., C. Foan and H. Fiedler (2003) PCB and PAH
releases from power stations and waste incineration processes in
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1
•)
•
The study suggests that ANT generates O2, O2 and OH
through Type I and Type II photosensitizing mechanisms at
ambient environmental intensities, which leads to linoleic acid
peroxidation, DNA damage, cell-cycle arrest and finally cell
damage. Results provide strong evidence that excess exposure
of ANT under sunlight may contribute to ANT mediated skin
phototoxicity ⁄ photomutagenesis in vivo. Since ANT photo-
degraded completely in 4 h sunlight exposure to its photo-
products, it is essential to investigate the phototoxic response
of its photoproducts to understand the total environmental
impact of ANT.
13. Wu, M. T., C. H. Pan, T. N. Wu, Y. L. Huang, C. Y. Chen, L. H.
Huang and C. K. Ho (2003) Immunological findings in a group of
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PLHC-1 cell line (Poeciliopsis lucida) and the role of lipid perox-
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toxicity and DNA damaging potential of benz(e)acephenanthryl-
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In conclusion, our study suggests that sunlight exposure
alters ANT to other forms. Sunlight and ANT induced a
transient p53 independent G1 arrest and expression of CYP
genes which may lead to photomutagenesis or photocarcino-
genesis. Human skin is exposed to solar radiation, therefore it
is important to investigate the human health hazards posed by
joint exposure of ANT and sunlight.
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and M. B. Greenberg (1997) Mechanistic quantitative structure–
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Yu and D. H. Saenz (2008) UVA photoirradiation of oxygenated
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Acknowledgements—The authors wish to thank the Director, IITR
for his support. We gratefully acknowledge the financial support
provided by SIP-08, CSIR, New Delhi, India.
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