Potential phototoxicity of rosuvastatin mediated by its dihydrophenanthrene-like photoproduct

Article abstract of DOI:10.1021/tx200341f

In this work, rosuvastatin has been used to gain insight into the molecular basis of statin photosensitization. This lipid-lowering drug, also known as "superstatin", contains a 2-vinylbiphenyl-like moiety and has been previously described to decompose under solar irradiation, yielding stable dihydrophenanthrene analogues. During photophysical characterization of rosuvastatin, only a long-lived transient at ca. 550 nm was observed and assigned to the primary photocyclization intermediate. Thus, the absence of detectable triplet-triplet absorption and the low yield of fluorescence rules out the role of the parent drug as an efficient sensitizer. In this context, the attention has been placed on the rosuvastatin main photoproduct (ppRSV). Indeed, the photobehavior of this dihydrophenanthrene-like compound presents the essential components needed for an efficient biomolecule photosensitizer i.e. (i) a high intersystem crossing quantum yield (Φ_ISC = 0.8), (ii) a triplet excited state energy of ca. 67 kcal mol^-1, and (iii) a quantum yield of singlet oxygen formation (Φ_Δ) of 0.3. Furthermore, laser flash photolysis studies revealed a triplet-triplet energy transfer from the triplet excited state of ppRSV to thymidine, leading to the formation of cyclobutane thymidine dimers, an important type of DNA lesion. Finally, tryptophan has been used as a probe to investigate the type I and/or type II character of ppRSV-mediated oxidation. In this way, both an electron transfer process giving rise to the tryptophanyl radical and a singlet oxygen mediated oxidation were observed. On the basis of the obtained results, rosuvastatin, through its major photoproduct ppRSV, should be considered as a potential sensitizer.

Full text of DOI:10.1021/tx200341f

ARTICLE
pubs.acs.org/crt
Potential Phototoxicity of Rosuvastatin Mediated by Its
Dihydrophenanthrene-like Photoproduct
Giacomo Nardi,† Virginie Lhiaubet-Vallet,*,† Paula Leandro-Garcia,‡ and Miguel Angel Miranda*,†
†Instituto de Tecnología Química UPV-CSIC, Universidad Politꢀecnica de Valencia, Avda de los Naranjos, s/n, 46022 Valencia, Spain
‡Organon/Merck, P.O. Box 20, 5340 B.H. Oss, The Netherlands
ABSTRACT: In this work, rosuvastatin has been used to gain insight into
the molecular basis of statin photosensitization. This lipid-lowering drug,
also known as “superstatin”, contains a 2-vinylbiphenyl-like moiety and
has been previously described to decompose under solar irradiation,
yielding stable dihydrophenanthrene analogues. During photophysical
characterization of rosuvastatin, only a long-lived transient at ca. 550 nm
was observed and assigned to the primary photocyclization intermediate.
Thus, the absence of detectable tripletꢀtriplet absorption and the low
yield of fluorescence rules out the role of the parent drug as an efficient
sensitizer. In this context, the attention has been placed on the rosuvas-
tatin main photoproduct (ppRSV). Indeed, the photobehavior of this
dihydrophenanthrene-like compound presents the essential components needed for an efficient biomolecule photosensitizer i.e.
(i) a high intersystem crossing quantum yield (ΦISC = 0.8), (ii) a triplet excited state energy of ca. 67 kcal molꢀ1, and (iii) a quantum
yield of singlet oxygen formation (ΦΔ) of 0.3. Furthermore, laser flash photolysis studies revealed a tripletꢀtriplet energy transfer
from the triplet excited state of ppRSV to thymidine, leading to the formation of cyclobutane thymidine dimers, an important type of
DNA lesion. Finally, tryptophan has been used as a probe to investigate the type I and/or type II character of ppRSV-mediated
oxidation. In this way, both an electron transfer process giving rise to the tryptophanyl radical and a singlet oxygen mediated
oxidation were observed. On the basis of the obtained results, rosuvastatin, through its major photoproduct ppRSV, should be
considered as a potential sensitizer.
’ INTRODUCTION
In addition, photolability of UVA/UVB-absorbing statins has been
shown to play a key role in their photosensitizing properties. In
fact, several members of this family have been reported to
decompose upon irradiation by a 6π electrocyclization process.8
Recently, the case of atorvastatin (ATV) has been studied in
detail, and it has been demonstrated that its phenanthrene-
like photoproduct behaves as an efficient singlet oxygen (1O2)
photosensitizer, giving rise to oxidation of biological components
like tryptophan or 20-deoxyguanosine.9 A study on ATV o-hydroxy
metabolite, which exhibits a slightly higher absorbance in the
UVB, has drawn similar conclusions.10 Finally, the phototoxic
potential of fluvastatin has been established by in vitro studies
with human keratinocytes and 3T3 fibroblasts. Again, its photo-
product, a benzocarbazole-like compound, has been revealed to
be a more efficient photosensitizer than the parent drug.11
In this work, the attention has been focused on rosuvastatin
(RSV, Chart 1), a synthetic statin of third generation also known
as “superstatin”. Photolysis of RSV has previously been studied
in the literature; it leads to formation of the stable dihydrophe-
nanthrene-like products (ppRSV and ppRSV3) and to the fully
conjugated ppRSV4 compound (Chart 1).8a Indeed, the photo-
reaction mechanism has been paralleled with that established for
Photosensitizing effects of xenobiotics are of increasing con-
cern in dermatology since modern lifestyle often combines
sunlight exposure with the presence of chemical substances in
the skin. In this context, an important number of compounds like
perfumes, sunscreen components or pharmaceuticals as nonster-
oidal anti-inflammatory agents,1 fluoroquinolone antibiotics2 or
phenothiazine neuroleptics3 have been reported for their photo-
sensitizing properties. Recently, the focus has been placed on
statin drugs, which are the most prescribed hypolipemiants in
Europe and in the USA.4 These lipid-lowering agents act in an
early and rate-limiting step of cholesterol biosynthesis by com-
petitive inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A
(HMG-CoA) reductase, an enzyme that catalyzes the conversion
of HMG-CoA to mevalonate. Among the adverse effects, several
clinical cases of cutaneous reaction have been reported and
associated with photosensivity disorders.5 Indeed, two different
mechanisms have been advanced to explain the molecular basis
of the statin sensitization process. The first one has been
proposed to account for the photosensitivity induced by lovas-
tatin, which in spite of its lack of absorbance above 280 nm has
been shown to be responsible for an enhancement of keratino-
cyte cellular damage under UVA irradiation.6 Indeed, this HMG-
CoA inhibitor produces an alteration of cell cholesterol content
that results in a higher sensitivity under UVA irradiation.6,7
Received: August 12, 2011
Published: September 16, 2011
r
2011 American Chemical Society
1779
dx.doi.org/10.1021/tx200341f Chem. Res. Toxicol. 2011, 24, 1779–1785
|

Chemical Research in Toxicology
ARTICLE
Chart 1. Structures of RSV and Its Photoproducts
to those previously reported.8a Although the two diastereoisomers
obtained can be chromatographically separated, the photophysical and
photobiological experiments were performed with the diastereoisomeric
mixture.
UVꢀVis Absorption Spectroscopy. Absorbances of the samples
were measured with a double beam Varian UVꢀvis model Cary 300
Scan spectrophotometer, using 1 cm pathway quartz cuvettes.
Laser Flash Photolysis (LFP). Two laser flash photolysis systems
were employed for the studies. For 355 nm excitation, experiments were
carried out using the fourth harmonic (λexc = 355 nm) of a Quantel
pulsed Nd:YAG spectrum laser system instrument. The single pulses
were ca. 10 ns duration, and the energy was ca.15 mJ/pulse. A pulsed
Excimer Laser Systems with Xe/HCl mixture was used for excitation at
308 nm. The single pulses were ∼17 ns duration, and the energy was
,100 mJ/pulse.
For both excitation systems, a xenon lamp was employed as the
detecting light source. The laser flash photolysis apparatus consisted of
the pulsed laser, the Xe lamp, a monochromator, and a photomultiplier
(PMT) system made up of side-on PMT, PMT housing, and a PMT
power supply. The output signal from the Tektronix oscilloscope was
transferred to a personal computer for study. All transient spectra were
recorded using 10 ꢁ 10 mm2 quartz cells with 4 mL capacity, and
solutions were bubbled for 10 min with N2, air, or O2, before acquisition.
The absorbance of the samples was kept in the range 0.30ꢀ0.40 at the
laser wavelength. Stock solutions of quenchers were prepared so that it
was only necessary to add microliter volumes to the sample cell to obtain
appropriate concentrations of the quencher. A linear quenching plot was
obtained, and the resulting rate constant was calculated from the slope of
the SternꢀVolmer plot:13
2-vinylbiphenyl compounds, where photochemical electro-
cyclization leads to an unstable 8a,9-dihydrophenanthrene
intermediate, which then undergoes a thermal sigmatropic
hydrogen shift to yield dihydrophenanthrene.8a,12 Due to the
photolability of RSV, the role of its photoproducts as photo-
sensitizing agents cannot be neglected. Here, the attention has
been focused on the main photoproduct: the dihydrophenan-
threne analogue ppRSV. Time-resolved and steady-state
experiments have been run to determine RSV and ppRSV
hotophysical properties, as well as their interaction with key
biomolecule building blocks, to investigate their potential role as
photosensitizers.
kobs ¼ k0 þ kq½Q ꢂ
where k0 is the triplet decay rate constant in the absence of quencher, kq
is the triplet decay rate constant in the presence of the quencher, and [Q]
is the quencher concentration in mol Lꢀ1. Photosensitization of ppRSV
triplet excited state by xanthone was performed using 355 nm excitation
wavelength, whereas determination of ppRSV triplet excited state
properties (lifetime, intersystem crossing quantum yield, interaction
with biological compounds and so on) was done exciting at 308 nm. In
the case of rosuvastatin LFP was also performed with a 308 nm laser.
A modified energy transfer methodology was used to determine the
molar absorption coefficient of tripletꢀtriplet transition of ppRSV
(ε(3ppRSV)). It consists of the quenching of xanthone triplet excited
state (λ = 633 nm) by ppRSV or 1-methylnaphthalene, used as standard.
Molar absorption coefficient of 3ppRSV was obtained from the ratio of
ΔA of ppRSV (at λ = 400 nm) and 1-methylnaphthalene (at λ = 415 nm)
when more than 95% of xanthone triplet was quenched. The following
equation was used:
’ EXPERIMENTAL PROCEDURES
Chemicals. Rosuvastatin was purchased from KEMPROTEC
Limited and was used without further purification. 2,2,6,6-Tetramethyl-
piperidine (TEMP) and β-carotene were purchased from Fluka.
Deuterated water (D2O), xanthone, carprofen, naphthalene (NP),
naproxen, perinaphthenone, L-tryptophan (Trp), thymidine (Thd),
1-methylnaphthalene (MeNP) and benzophenone (BP) were from
Sigma Aldrich.
The reagent grade solvents were obtained from Scharlau and used
without further purification. Phosphate buffered saline (PBS) solutions
of 0.01 M (pH = 7.4) were prepared by dissolving tablets (Sigma-
Aldrich) in Milli-Q water.
Photoreactors. Irradiations were performed by means of a multi-
lamp photoreactor equipped with lamps with a maximal output at ca.
300 nm (Hitachi, F15T8/BL). In the case of monochromatic irradiation,
a Microbeam system (model L-201), including a 150 W xenon lamp
coupled with a monochromator (model 101), was used.
Production of ppRSV. Compound ppRSV was isolated as de-
scribed in the literature.8a Briefly, a dispersion of rosuvastatin calcium
(75 mg in 150 mL of water) was irradiated for 20 min in Pyrex tubes
using the above-described photoreactor equipped with six lamps. The
irradiation mixture was dried under vacuum and separated by silica gel
thin-layer chromatography eluting with CH2Cl2/MeOH (95:5) with
drops of acetic acid (two runs) to obtain the diastereometric mixture of
ppRSV. All of the experiments were conducted at room temperature and
under air atmosphere. The 1H and 13C NMR spectral data were identical
3
3
εð ppRSVð400ÞÞ ¼ εð MeNPð415ÞÞ
3
3
ꢁ ΔAð ppRSVð400ÞÞ=ΔAð MeNPð415ÞÞ
where ΔA(3ppRSV(400)) and ΔA(3 MeNP (415)) refer to the transient
absorption of ppRSV and 1-methylnaphthalene triplet state (at 400 and
415 nm, respectively) at the end of the process, and ε(3 MeNP(415))
corresponds to the known molar absorption coefficient of the 1-methyl-
naphthalene triplet at 415 nm (ε = 11 200 Mꢀ1 cmꢀ1 in acetonitrile).14
Then, intersystem quantum yield (ΦISC) was determined by the
comparative method taking into account the amount of ppRSV and
naphthalene populated after 308 nm laser excitation. Isoabsortive
degassed acetonitrile solutions of both compounds were used. So, ΦISC
3
1780
dx.doi.org/10.1021/tx200341f |Chem. Res. Toxicol. 2011, 24, 1779–1785

Chemical Research in Toxicology
ARTICLE
Figure 2. Transient absorption spectra of RSV (4.8 ꢁ 10ꢀ5 M) in PBS
solution, recorded using a fresh solution for each laser pulse (Xe/Cl
excimer, 308 nm excitation).
Figure 1. Absorption spectra of RSV 4.8 ꢁ 10ꢀ5 M (9) and ppRSV 1.5 ꢁ
10ꢀ5 M (O) in PBS.
was obtained by using the following equation:
3
3
3
EPR Spin Trapping Experiments. The measurements were
performed in a flat cell with a Bruker EMX 10/12 EPR spectrometer,
using the following parameters: microwave power, 20 mW; modulation
amplitude, 1.0 G; and modulation frequency, 100 kHz. Analysis was
performed recording the EPR signal of the free radical TEMPO
generated by reaction of singlet oxygen with the spin trap TEMP.17
Aerated methanolic solutions of 10 mM TEMP containing ppRSV, with
an absorbance of ∼0.3 at 300 nm, were irradiated in a photoreactor; EPR
spectra were recorded at different irradiation times.
Tryptophan Photodegradation. PBS solution of ppRSV (2 ꢁ
10ꢀ5 M) containing an equimolar concentration of tryptophan was
irradiated at 320 nm using a MicroBeam system. Tryptophan fluores-
cence emission intensity (λex = 282 nm, λem = 353 nm) was recorded at
increasing irradiation time. A parallel experiment was performed using
deuterium oxide as the solvent under identical conditions. A solution of
tryptophan in PBS (2 ꢁ 10ꢀ5 M) was used as control.
ΦISCð ppRSVÞ ¼ ΦISCð NPÞ ꢁ ΔAð ppRSVð400ÞÞ
3
3
3
ꢁεð NPð420Þ=½ΔAð NPð420ÞÞ ꢁ εð ppRSVð400Þꢂ
where ΦISC (NP) corresponds to the intersystem crossing quantum
yield of naphthalene; ε(3NP(420)) refers to the molar absorption
coefficient of naphthalene triplet at 420 nm; ΔA(3ppRSV (400)) and
3
ΔA(NP(415)) refer to the transient absorption of ppRSV and NP
triplet at 400 and 520 nm, respectively. The values of ε(3NP(420)) and
ΦISC(NP) in acetonitrile were taken as 15 000 Mꢀ1 cmꢀ1 and 0.8,
respectively.15
Singlet Oxygen Measurements. The singlet oxygen phosphor-
escence decay traces after the laser pulse were registered at 1270 nm
employing a Peltier-cooled (ꢀ62.8 °C) Hamamatsu NIR detector
operating at 588 V, coupled to a computer-controlled grating mono-
chromator. A pulsed Nd:YAG L52137 V LOTIS TII was used at the
excitation wavelength of 266 nm. The single pulses were ca. 10 ns
duration, and the energy was lower than 10 mJ/pulse. The laser flash
photolysis system consisted of the pulsed laser, a 77250 Oriel mono-
chromator and an oscilloscope DP04054 Tektronix. The output signal
from the oscilloscope was transferred to a personal computer. All
measurements were made at room temperature, air atmosphere, using
acetonitrile as solvents in 1 cm pathway quartz cuvettes. The absorbance
of the samples was 0.30 at the laser wavelength. The singlet oxygen
quantum yield (ϕΔ) was determined using naproxen in acetonitrile
(ϕΔ = 0.27)1 as standard. Singlet oxygen formation was calculated from
the slope of the plots of signal intensity at zero time versus laser light
intensity according to the following equation:
’ RESULTS
Photophysics of Rosuvastatin. Rosuvastatin absorption
reaches the UVA region of the spectrum; it exhibits a maximum
at 243 nm and a shoulder until 350 nm (Figure 1). A very weak
fluorescence emission was observed at 373 nm (data not shown).
This emission was increasing during measurement of successive
spectra with the same sample, suggesting degradation of the
compound under UV irradiation.
Photolability was further observed by laser flash photolysis
(LFP) experiments performed on RSV N2-degassed PBS solu-
tions. Indeed, after one laser pulse, the decay monitored at
400 nm exhibited a remarkable change, suggesting formation of a
new intermediate. A transient absorption spectrum of RSV,
obtained using a fresh sample for each point, showed a long-
lived species centered at ca. 550 nm (Figure 2). By comparison
with literature data available for 2-vinylbiphenyl derivatives,12
this transient has been assigned to the intermediate of cyclization
of RSV leading to the dihydrophenanthrene-like compounds.
When LFP of an irradiated sample was run, a signal with
maximum at 400 nm was detected. This species was quenched by
oxygen, and thus it was tentatively assigned to the triplet excited
state of RSV photoproduct(s). With this background, in view of
the important photolability of the parent drug, the attention was
centered on the photobehavior of the primary and main photo-
product, namely, ppRSV.
Isample
ϕΔ
¼
ϕΔ
ðstandardÞ
ðsampleÞ
Istandard
where Isample is the emission intensity for the sample, Istandard is the
emission intensity for the standard and ϕΔ(standard) is the standard
quantum yield of singlet oxygen formation.
Fluorescence. The steady-state fluorescence experiments were
carried out on a Photon Technology International (PTI) LPS-220B
spectrofluorometer. Fluorescence quantum yield measurements were
performed with aerated methanol, acetonitrile and PBS solutions;
the absorbance was adjusted to ca. 0.1 at the excitation wavelength (at
λex = 314 nm), and carprofen was used as standard (ΦF = 0.068
in acetonitrile).16 All measurements were recorded using 1 cm path-
way quartz cells with 4 mL capacity and were carried out at room
temperature.
1781
dx.doi.org/10.1021/tx200341f |Chem. Res. Toxicol. 2011, 24, 1779–1785

Chemical Research in Toxicology
ARTICLE
Figure 3. Fluorescence excitation (O, λem = 376 nm) and emission
(9, λexc = 314 nm) of ppRSV in acetonitrile at 295 K. Phosphorescence
emission spectrum in EtOH at 77 K (Δ, λexc = 312 nm).
Figure 4. Transient spectrum of tripletꢀtriplet transition obtained for a
degassed acetonitrile solution of ppRSV (3.3 ꢁ 10ꢀ5 M) after 308 nm
laser excitation.
Table 1. Photophysical Properties of ppRSV in Different
Solvents
λem
ES
λTꢀT
τT
kq (O2)
(nm) (kcal molꢀ1
)
ΦF (nm) (μs) (Mꢀ1 sꢀ1
)
PBS
368
370
84
84
84
0.036 400
0.012 400
0.014 400
20
10
8
1.2 ꢁ 109
methanol
2.6 ꢁ 109
2 ꢁ 109
acetonitrile 376
Photophysics of the Major Rosuvastatin Photoproduct. As
shown in Figure 1, ppRSV exhibits a defined absorption band
centered at λmax = 313 nm. It is noteworthy that the photo-
product has a stronger absorption in the UVA region than RSV,
which thus increases its potential to act as a photosensitizer under
solar irradiation. Steady-state fluorescence of ppRSV in acetoni-
trile solution showed an emission spectrum with λmax at 376 nm
and a fluorescence quantum yield ΦF of 0.014, obtained using
carprofen as standard. Fluorescence emission was not quenched
by oxygen. A singlet-state energy (ES) of 84 kcal molꢀ1 was
determined by considering the intersection between the normal-
ized emission and excitation spectra (Figure 3). Similar results
were obtained using PBS and methanol as solvent (Table 1). Low
temperature emission was also performed in EtOH at 77 K.
Under these conditions, ppRSV showed a well-defined emission
spectrum with a maximum centered at 460 nm. A triplet excited
state energy value (ET(ppRSV)) of 67 kcal molꢀ1 was obtained
from the 0ꢀ0 band of the spectrum.
Figure 5. LFP at 355 nm excitation. Decays of xanthone (6.6 ꢁ 10ꢀ5 M
in deaerated acetonitrile) monitored at 630 nm, in the presence of
increasing concentration of ppRSV (from 0 to 6.6 ꢁ 10ꢀ5 M). Inset:
SternꢀVolmer plot.
tripletꢀtriplet energy transfer is occurring, which agrees with the
triplet state nature of the 400 nm transient species. A comple-
mentary experiment was also performed; it consisted of the
quenching of the triplet excited state of a donor using ppRSV as
3
acceptor. Taking into account the ppRSV energy value deter-
mined above, xanthone was used as a suitable photosensitizer (ET
= 74.2 kcal molꢀ1).15 As shown in Figure 5, xanthone tripletꢀ
triplet absorption centered at 630 nm was quenched in the
presence of ppRSV with a bimolecular rate constant of 1.6 ꢁ 1010
Laser flash photolysis of deaerated solutions of ppRSV showed
a transient at 400 nm (Figure 4) with lifetime varying from 8 μs in
methanol to 20 μs in PBS. This species was efficiently quenched
by oxygen, and thus it was tentatively assigned to ppRSV triplet
excited state (3ppRSV) (Table 1). Interestingly, this signal was
similar to that observed during LFP of an irradiated sample of
RSV (see above).
M
ꢀ1 sꢀ1, thus confirming on the one hand the triplet nature of
the 400 nm transient and on the other hand that ET (3ppRSV) is
lower than ET (xanthone).
3
Further characterization of ppRSV was performed by deter-
The nature of ppRSV transient was confirmed by further
quenching experiments. In a first stage, a diffusion-controlled
process was observed in the presence of β-carotene (ET ca.
20 kcal molꢀ1).15 Under these conditions, the transient absorp-
tion at 400 nm disappeared concomitantly with the appear-
ance of a new band peaking at 520 nm, which corresponds
to the characteristic TꢀT absorption of β-carotene. Thus, a
mining the molar absorption coefficient of its tripletꢀtriplet
absorption in acetonitrile. The signals formed after quenching of
xanthone triplet excited state by ppRSV or 1-methylnaphthalene
were compared, using the latter as standard (see Experimental
Procedures). In this way, a value of 8500 Mꢀ1 cmꢀ1 at 400 nm was
obtained, and the intersystem crossing quantum yield (ΦISC) was
estimated at 0.8 by means of the comparative method.
1782
dx.doi.org/10.1021/tx200341f |Chem. Res. Toxicol. 2011, 24, 1779–1785

Chemical Research in Toxicology
ARTICLE
Figure 6. TEMPO signals obtained by irradiation of a methanolic
solution of TEMP (10 mM) and ppRSV (3.3 ꢁ 10ꢀ5 M): without
irradiation (0) and after 5 (2) and 7 (O) min of irradiation.
Figure 7. LFP at 308 nm excitation of ppRSV (3.3 ꢁ 10ꢀ5 M) in
deaerated PBS, transient decay monitored at 400 nm without (9) and
with 10ꢀ3 M Thd (O). Inset: SternꢀVolmer plot.
Singlet Oxygen Generation. Singlet oxygen (1O2) is one of
the main reactive oxygen species (ROS) involved in the oxidative
damage to living systems. It is now well-established that O2,
1
In the first stage, interaction with DNA components was
considered by monitoring the ppRSV decay at 400 nm in the
3
photogenerated by endogenous or exogenous compounds, is able
to react with a large number of target biomolecules, including
unsaturated lipids, proteins, and nucleic acids. The efficient
3ppRSV quenching by molecular oxygen observed by LFP should
giverise to the formation of 1O2 bya tripletꢀtriplet energytransfer
process. To confirm production of this species, spin trapping
experiments were conducted using the well-known conversion of
TEMP to the stable free radical TEMPO, detectable by EPR
analysis. A methanolic solution of TEMP was irradiated in the
presence of ppRSV. As shown in Figure 6, under these conditions,
detection and increase of the typical TEMPO signal (g = 2.0054,
aN = 16.3 G) as a function of irradiation time provided clear
evidence for the role of ppRSV as singlet oxygen sensitizer.
The quantum yield of 1O2 formation was determined by time-
resolved near-IR emission studies using naproxen as standard
(ΦΔ = 0.27 in acetonitrile).1 The intensity of the characteristic
singlet oxygen emission at 1270 nm was plotted as a function of
the laser intensity for oxygenated acetonitrile solutions of ppRSV
and naproxen. A quantum yield of 0.3 was obtained from the ratio
of the linear plot slopes. The same value was obtained by
registering steady-state emission of 1O2, using perinaphthenone
as standard (ΦΔ = 1 in acetonitrile).18
presence of increasing amounts of Thd. As shown in Figure 7,
addition of the nucleoside results in the shortening of ppRSV
3
lifetime. A bimolecular quenching rate constant of 3 ꢁ 107 Mꢀ1 sꢀ1
was determined from the SternꢀVolmer plot (Figure 7,
inset). Despite the relatively low value of this quenching con-
stant, tripletꢀtriplet energy transfer between 3ppRSV and Thd,
which reflects the potential Thd<>Thd dimer formation, is
feasible. Moreover, the obtained value is in agreement with
expectations from the isoenergetic states of the donor and the
acceptor.20
Related studies were also performed with a protein building
block. Specifically, tryptophan (Trp) represents an interesting
probe as it can be oxidized both by type I and type II mechanisms.
3
In this context, the oxidizing potential of ppRSV has been
evaluated by means of time-resolved and steady-state experi-
ments. Direct interaction of 3ppRSV with Trp has been studied
by LFP, and quenching by this amino acid (Figure 8A) was found
to occur with a bimolecular rate constant of 5 ꢁ 109 Mꢀ1 sꢀ1
,
leading to formation of the long-lived tryptophanyl radical (λmax
at 310 and 520 nm).21 This is in agreement with a mechanism
3
involving electron transfer from Trp to ppRSV, followed by
deprotonation of the resulting radical cation at pH = 7.4, to form
Interaction with Biomolecules. After showing that ppRSV
exhibits the appropriate properties of an efficient biomolecule
the tryptophanyl radical.21
In addition, the ppRSV photoreactivity was investigated by
steady-state photolysis, by irradiation of ppRSV at 320 nm in the
presence of equimolar amounts of Trp. The disappearance of the
amino acid fluorescence emission (λmax = 353 nm) with irradia-
tion time was observed and taken as measurement of the extent
of oxidation (Figure 8B). To check the involvement of a type II
mechanism, a parallel experiment was performed in D2O, where
the lifetime of 1O2 is longer than in water. As a matter of fact, the
reaction rate was increased by a factor of 2 under these condi-
tions, in agreement with Trp oxidation by 1O2.
3
photosensitizer, the next step was to check whether ppRSV
might be involved in oxidation of biological substrates. In this
context, the type I mechanism proceeds via hydrogen abstraction
or electron transfer mechanisms, whereas the type II oxidation is
1
mediated by the formation and subsequent reactions of O2.19
Both mechanisms can be in the origin of protein or DNA
photosensitization. In addition, a tripletꢀtriplet energy transfer
from a photosensitizer to thymidine (Thd) could give rise to
thymidine cyclobutane dimers (Thd<>Thd), an important DNA
lesion.20 As a requirement for this process to occur, the triplet
state energy of the photosensitizer has to be higher than that of
the DNA base. In this context, the ET value of 3ppRSV,
determined as 67 kcal molꢀ1, is high enough to make this
compound a potential Thd<>Thd photosensitizer.
’ DISCUSSION
Nanosecond LFP of the statin drug RSV reveals a long-
lived transient at λmax ca. 550 nm, which was assigned to
1783
dx.doi.org/10.1021/tx200341f |Chem. Res. Toxicol. 2011, 24, 1779–1785

Chemical Research in Toxicology
ARTICLE
Scheme 1
determined by phosphorescence emission studies. Indeed, triplet
excited states have been demonstrated to be crucial species in
photosensitizing reactions. 1,16 Thus, in connection with ppRSV
photophysical properties, three processes have been considered
to investigate the interaction between 3ppRSV and biomolecules
(eqs 5ꢀ8, Scheme 1).
Thymidine dimers (Thd<>Thd) are the most important
lesions formed upon direct irradiation of DNA; however,
they can also be obtained during photosensitization by a well
established process based on a tripletꢀtriplet energy transfer.20
Few compounds are known to exhibit the appropriate properties
to act as efficient sensitizers, the requirement being mainly based
on the ET value. Here, the 3ppRSV triplet excited state lies close
to that of benzophenone or carprofen, reported as Thd<>Thd
3
photosensitizer.20 As a matter of fact, quenching of ppRSV
in the presence of Thd is in agreement with a tripletꢀtriplet
energy transfer process (eq 6, Scheme 1). As expected from the
isoenergetic triplet excited states of the donor and the acceptor,
the reaction occurs with a low bimolecular rate constant of ca.
Figure 8. (A) LFP at 308 nm excitation of ppRSV (3.3 ꢁ 10ꢀ5 M) in
deaerated PBS, transient decay monitored at 400 nm without (9) and
with 5 ꢁ 10ꢀ6 M (Δ) Trp. Inset: Transient absorption spectrum of
ppRSV in the presence of 1.8 ꢁ 10ꢀ5 M Trp registered 10.5 μs after the
laser pulse. (B) Tryptophan photodegradation. Normalized emission at
λem = 353 nm monitored as a function of irradiation time: Trp alone in
PBS (9), and in the presence of equimolar amounts of Trp and ppRSV
(2 ꢁ 10ꢀ5 M) in PBS (O) and in deuterium oxide as solvent (Δ).
107 Mꢀ1 sꢀ1
.
Furthermore, oxidations represent an essential part of bio-
molecule photodamages. Here, tryptophan has been used as a
probe to determine type I and/or type II character of ppRSV
mediated photooxidation. Steady-state photolysis has shown a
higher degradation of this amino acid in D2O, pointing toward
a 1O2 mediated oxidation. This result is in accordance with the
obtained ppRSV singlet oxygen quantum yield ΦΔ of 0.3 (eq 5,
Scheme 1). In addition, a type I reactivity has also been
8a,9-dihydrophenanthrene intermediate.12 This result agrees well
with RSV photochemistry corresponding to an electrocyclization
process followed by a sigmatropic H transfer to form the primary
photoproduct ppRSV.8a Moreover, the lack of triplet excited
state detection and the low fluorescence emission indicate that
RSV should be considered as an unlikely photosensitizer, as
previously reported for ATV.9 In the last case, we demonstrated
that a photoproduct rather than the parent drug is the origin of
the photosensitized damage to biomolecules.
With this background, the attention has been placed on
ppRSV. It is noteworthy that its dihydrophenanthrene-like
structure is the origin of interesting photophysical properties
with respect to the phenanthrene-like photoproduct of ATV.
Indeed, the photosensitizing capacity of this latter is mainly
governed by singlet oxygen reactivity.9
As shown in Scheme 1, after promotion to its singlet excited
state (eq 1, Scheme 1), ppRSV can undergo different deactiva-
tion pathways, including fluorescence (eq 2) and intersystem
crossing (eq 3). Based on LFP experiments, an efficient triplet
excited state formation (ΦISC = 0.8) has been established and
consequently a high potential as a photosensitizer. Moreover, a
high triplet state energy (ET ca. 67 kcal molꢀ1) state has been
3
evidenced by LFP. Indeed, ppRSV quenching by Trp (eq 7,
Scheme 1) leads to formation of the tryptophanyl radical
arising from an electron transfer process. Therefore, oxygen
and Trp compete for 3ppRSV quenching. The predominance of
type I or type II mechanism can be inferred from the absolute
quenching rates (kq ꢁ [Q]) that consider not only the bimo-
lecular rate constant kq but also the concentration of the
quencher [Q]. Thus, considering the maximum quencher
concentration used in this work (i.e. 1.27 ꢁ 10ꢀ3 M and
20 ꢁ 10ꢀ6 M for oxygen and Trp, respectively), a type II/type
I ratio of 14 is obtained. Although this value only reflects the
competition between processes (5) and (7) of Scheme 1 and
does not take into account the subsequent steps, it clearly
indicates that Trp is mainly decomposed by a singlet oxygen
process.
In summary, it has been demonstrated that RSV, a photolabile
UV-absorbing statin, presents the appropriate properties of an
efficient photosensitizer, acting through photoproduct forma-
tion. This may be the origin of undesired side effects like
phototoxicity and/or photogenotoxicity.
1784
dx.doi.org/10.1021/tx200341f |Chem. Res. Toxicol. 2011, 24, 1779–1785

Chemical Research in Toxicology
ARTICLE
’ AUTHOR INFORMATION
exposure of the drug rosuvastatin in water. J. Photochem. Photobiol., A
187, 263–268. (b) Grobelny, P., Viola, G., Vedaldi, D., Dall’Acqua, F.,
Gliszczynska-Swiglo, A., and Mielcarek, J. (2009) Photostability of
pitavastatin-A novel HMG-CoA reductase inhibitor. J. Pharm. Biomed.
Anal. 50, 597–601. (c) Cermola, F., DellaGreca, M., Iesce, M. R.,
Montanaro, S., Previtera, L., and Temussi, F. (2006) Photochemical
behavior of the drug atorvastatin in water. Tetrahedron 62, 7390–7395.
(d) Cermola, F., DellaGreca, M., Iesce, M. R., Montanaro, S., Previtera,
L., Temussi, F., and Brigante, M. (2007) Irradiation of fluvastatin in
water: Structure elucidation of photoproducts. J. Photochem. Photobiol.,
A 189, 264–271.
Corresponding Author
*V.L.-V.: Instituto de Tecnología Química UPV-CSIC, Universidad
Politꢀecnica de Valencia, Avda de los Naranjos, s/n, 46022
Valencia, Spain; tel, +34-963879697; fax, +34-963877809;
e-mail, lvirgini@itq.upv.es. M.A.M.: Instituto de Tecnología
Química UPV-CSIC, Universidad Politꢀecnica de Valencia, Avda
de los Naranjos, s/n, 46022 Valencia, Spain; tel, +34-963877807;
fax, +34-963877809; e-mail, mmiranda@qim.upv.es.
(9) Montanaro, S., Lhiaubet-Vallet, V., Iesce, M., Previtera, L., and
Miranda, M. A. (2008) A mechanistic study on the phototoxicity of
atorvastatin: singlet oxygen generation by a phenanthrene-like photo-
product. Chem. Res. Toxicol. 22, 173–178.
Funding Sources
Spanish Government (CTQ2009-13699, RyC-2007-00476, PFIS
FI09/00312) is gratefully acknowledged for financial support.
(10) Alarcon, E., Gonzalez-Bejar, M., Gorelsky, S., Ebensperger, R.,
Lopez-Alarcon, C., Netto-Ferreira, J. C., and Scaiano, J. C. (2010)
Photophysical characterization of atorvastatin (Lipitor) ortho-hydroxy
metabolite: role of hydroxyl group on the drug photochemistry. Photo-
chem. Photobiol. Sci, 9, 1378–1384.
’ ACKNOWLEDGMENT
We thank Dr. Sara Montanaro for preliminary experiments.
(11) Viola, G., Grobelny, P., Linardi, M. A., Salvador, A., Basso, G.,
Mielcarek, J., Dall’Acqua, S., Vedaldi, D., and Dall’Acqua, F. (2010)
The phototoxicity of fluvastatin, an HMG-CoA reductase inhibitor,
is mediated by the formation of a benzocarbazole-like photoproduct.
Toxicol. Sci. 118, 236–250.
’ ABBREVIATIONS
ATV, atorvastatin; BP, benzophenone; LFP, laser flash photo-
lysis; MeNP, 1-methylnaphthalene; NP, naphthalene; ppRSV,
rosuvastatin photoproduct; RSV, rosuvastatin
(12) (a) Fornier de Violet, P., Bonneau, R., Lapouyade, R., Koussini,
R., and Ware, W. R. (1978) Intramolecular photocyclization of 2-vinyl-
biphenyl-like compounds. 2. Detection of the intermediates and kinetic
study by laser flash photolysis of 1-(o-diphenyl)-1-phenylethylene.
J. Am. Chem. Soc. 100, 6683–6687. (b) Lewis, F. D., and Zuo, X.
(2003) Conformer-specific photoisomerizaton of some 2-vinylbiphe-
nyls. Photochem. Photobiol. Sci. 2, 1059–1066.
’ REFERENCES
(1) Bosca, F., Marin, M. L., and Miranda, M. A. (2001) Photoreac-
tivity of the nonsteroidal anti-inflammatory 2-arylpropionic acids with
photosensitizing side effects. Photochem. Photobiol. 74, 637–655.
(2) Lhiaubet-Vallet, V., Bosca, F., and Miranda, M. A. (2009)
Photosensitized DNA damage: the case of fluoroquinolones. Photochem.
Photobiol. 85, 861–868.
(3) Viola, G., and Dall’Acqua, F. (2006) Photosensitization of
biomolecules by phenothiazine derivatives. Curr. Drug Targets 7,
1135–1154.
(4) Istvan, E. S., and Deisenhofer, J. (2001) Structural mechanism
for statin inhibition of HMG-CoA reductase. Science 292, 1160–1164.
(5) (a) Granados, M. T. R., de la Torre, C., Cruces, M. J., and Pineiro,
G. (1998) Chronic actinic dermatitis due to simvastatin. Contact
Dermatitis 38, 294–295. (b) Holme, S. A., Pearse, A. D., and Anstey,
A. V. (2002) Chronic actinic dermatitis secondary to simvastatin.
Photodermatol., Photoimmunol. Photomed. 18, 313–314. (c) Marguery,
M. C., Chouini-Lalanne, N., Drugeon, C., Gadroy, A., Bayle, P., Journe,
F., Bazex, J., and D’Incan, M. (2006) UV-B phototoxic effects induced by
atorvastatin. Arch. Dermatol. 142, 1082–1084. (d) Morimoto, K.,
Kawada, A., Hiruma, A., Ishibashi, A., and Banba, H. (1995) Photo-
sensitivity to simvastatin with an unusual response to photopatch
and phototests. Contact Dermatitis 33, 274. (e) Roger, D., Rolle, F.,
Labrousse, F., Brosset, A., and Bonnetblanc, J. M. (1994) Simvastatin-
induced lichenoid drug eruption. Clin. Exp. Dermatol. 19, 88–89.
(f) Rodríguez-Pazos, L., Sꢀanchez-Aguilar, D., Rodríguez-Granados, M.,
Pereiro-Ferreirꢀos, M., and Toribio, J. (2010) Erythema multiforme
photoinduced by statins. Photodermatol., Photoimmunol. Photomed.
26, 216–218. (g) Del Rio, R., Palou, J., and Lecha, M. (1998)
Fotosensibilidad por estatinas. Boletin GEF 32.
(13) Stern, O., and Volmer, M. (1919) The fading time of fluores-
cence. Phys. Z. 20, 183–188.
(14) Carmichael, I., and Hug, G. L. (1986) Triplet-triplet absorption
spectra of organic molecules in condensed phases. J. Phys. Ref. Data 15, 1.
(15) Montalti, M., Credi, A., Prodi, L., and Gandolfi, M. T. (2006)
Handbook of photochemistry, 3rd ed., CRC Press, Boca Raton, FL.
(16) Bosca, F., Encinas, S., Heelis, P. F., and Miranda, M. A. (1997)
Photophysical and photochemical characterization of a photosensitizing
drug: a combined steady state photolysis and laser flash photolysis study
on carprofen. Chem. Res. Toxicol. 10, 820–827.
(17) Lion, Y., Delmelle, M., and Van de Vorst, A. (1976) New
method of detecting singlet oxygen production. Nature 263, 442–446.
(18) Schmidt, R., Tanielian, C., Dunsbach, R., and Wolff, C. (1994)
Phenalenone, a universal reference compound for the determination of
quantum yields of singlet oxygen O2(1Δg) sensitization. J. Photochem.
Photobiol., A 79, 11–17.
(19) Foote, C. S. (1991) Definition of type-I and type-II photo-
sensitized oxidation. Photochem. Photobiol. 54, 659–659.
(20) Cuquerella, M. C., Lhiaubet-Vallet, V., Bosca, F., and Miranda,
M. A. (2011) Photosensitised pyrimidine dimerisation in DNA. Chem.
Sci. 2, 1219–1232.
(21) Tsentalovich, Y. P., Snytnikova, O. A., and Sagdeev, R. Z.
(2004) Properties of excited states of aqueous tryptophan. J. Photochem.
Photobiol., A 162, 371–379.
(6) Quiec, D., Maziere, C., Auclair, M., Santus, R., Gardette, J.,
Redziniak, G., Franchi, J., Dubertret, L., and Maziere, J. C. (1995)
Lovastatin enhances the photocytotoxicity of UVA radiation towards
cultured N.C.T.C. 2544 human keratinocytes: prevention by cholesterol
supplementation and by a cathepsin inhibitor. Biochem. J. 310, 305–309.
(7) Chignell, C. F., Kukielczak, B. M., Sik, R. H., Bilski, P. J., and He,
Y.-Y. (2006) Ultraviolet A sensitivity in Smith-Lemli-Opitz syndrome:
Possible involvement of cholesta-5,7,9(11)-trien-3[beta]-ol. Free Radi-
cal Biol. Med. 41, 339–346.
(8) (a) Astarita, A., DellaGreca, M., Iesce, M. R., Montanaro, S.,
Previtera, L., and Temussi, F. (2007) Polycyclic compounds by sunlight
1785
dx.doi.org/10.1021/tx200341f |Chem. Res. Toxicol. 2011, 24, 1779–1785