UVA photoinduced yeast protein modifications by methylene blue and naproxen

Article abstract of DOI:10.1039/c3pp25359g

UVA photosensitization by methylene blue (MB) or by naproxen (NAP) towards cell proteins in yeast Saccharomyces cerevisiae was investigated in order to compare this system with two simpler models, such as free Trp in solution and as a component of bovine and human serum albumin. The process was studied by monitoring protein tryptophan (Trp) residue integrity. The sensitized photodegradation of proteins resulted in different degrees of Trp damage with different Trp (photo)-products. Indeed, many of these Trp derivatives are diagnostic for the photosensitization mechanism and some of them were obtained from cells by UVA photosensitization for the first time in this work. The analysis of quantum yields of photoproduct distribution allowed us to weigh up the type I/II contribution on a UVA photosensitization mechanism. The UVA mediated generation of these Trp derivatives is consistent with the occurrence of singlet oxygen formation (almost dominant in MB), and photoionization (significant in NAP) within the protein matrix. The results obtained in the case of this more complex system (cell) are in agreement with the two simpler models recently studied in our lab. The quantum yields of Trp photoinduced degradation, as well as of its photoproducts formation, decrease with increasing the complexity of the investigated target. The Royal Society of Chemistry and Owner Societies 2013.

Full text of DOI:10.1039/c3pp25359g

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Photobiological Sciences
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UVA photoinduced yeast protein modifications
by methylene blue and naproxen
Cite this: Photochem. Photobiol. Sci., 2013,
2, 967
1
a
a
a,b
Giuseppina Bracchitta, Alfio Catalfo and Guido De Guidi*
Received 26th October 2012,
Accepted 8th March 2013
DOI: 10.1039/c3pp25359g
www.rsc.org/pps
UVA photosensitization by methylene blue (MB) or by naproxen photosensitization considers some drugs that when taken sys-
NAP) towards cell proteins in yeast Saccharomyces cerevisiae was temically or injected intravitreally can be phototoxic for eye
(
3
4,5
investigated in order to compare this system with two simpler lens proteins and may contribute to early cataractogenesis.
models, such as free Trp in solution and as a component of bovine In proteins, the main photooxidation targets are the amino
and human serum albumin. The process was studied by monitor- acids tryptophan (Trp), tyrosine, phenylalanine, and cysteine/
6
ing protein tryptophan (Trp) residue integrity. The sensitized cystine. Photooxidation can lead also to skin aging and dis-
2
6
photodegradation of proteins resulted in different degrees of Trp eases, as well as yellowing in wool fabric. These events are
damage with different Trp (photo)-products. Indeed, many of mediated by a variety of peroxides (hydroperoxides and endo-
these Trp derivatives are diagnostic for the photosensitization peroxides), which can subsequently react with photogenerated
7
mechanism and some of them were obtained from cells by UVA free radicals. Unfortunately, the photosensitized reactions of
photosensitization for the first time in this work. The analysis of proteins turn out to be very complex, which usually dismisses
8
quantum yields of photoproduct distribution allowed us to weigh attempts to establish their course.
up the type I/II contribution on a UVA photosensitization mechan-
Methylene blue [3,7-bis(dimethylamino)-phenazathionium
9
ism. The UVA mediated generation of these Trp derivatives is con- chloride] (MB) is a good model of type II photosensitizer,
sistent with the occurrence of singlet oxygen formation (almost because its most important mechanistic aspect is to promote
9
dominant in MB), and photoionization (significant in NAP) within photooxidative reactions mainly by singlet oxygen (E
T
=
the protein matrix. The results obtained in the case of this more 142.1 kJ mol− , Φ = 0.60 in water ) whereas MB photoioniza-
1
10
Δ
complex system (cell) are in agreement with the two simpler tion is a minor process (Φe− = 5.0 × 10− in water ). Thus, as
models recently studied in our lab. The quantum yields of Trp shown by the low quantum yield of solvated electrons, the type
photoinduced degradation, as well as of its photoproducts for- I reaction is negligible. MB acts as a photosensitizer towards
3
11
1
2–15
mation, decrease with increasing the complexity of the investi- various targets, including proteins.
Visible light induced
gated target.
MB photolysis occurs by low yield multiphotonic processes
1
1
involving solvated electrons but the main photosensitization
1
6
pathway occurs by singlet oxygen generation. MB can thus be
chosen as a suitable type II UVA-VIS sensitizer on protein Trp.
In consequence, it can be used for a comparison with other
Introduction
Photosensitizers, when irradiated, can induce different altera- photosensitizers.
tions in biomolecules (e.g., proteins, nucleic acids, unsaturated
Among them, naproxen (NAP), 2-(6-methoxy-2-naphthyl)-
lipids, and other cell constituents) causing phototoxic propionic acid, is a non-steroidal anti-inflammatory drug
1
7
phenomena such as photoallergy and loss of key biological (NSAID). This class of drugs induces photosensitization on
1
18,19
functions (inactivation of enzymes, hormones, etc.). Moreover, different targets,
in particular in protein photo-cross-
2
0
21
proteins, porphyrins, flavins and vitamins can act as endogen- linking, responsible for photoallergic and phototoxic reac-
2
2,23
ous skin sensitizer chromophores and are emerging as key tions.
Laser flash photolysis studies evidenced that during
specific case of NAP photolysis an excited triplet is involved. The excited
state undergoes two processes: energy transfer to molecular
2
24
intermediates of skin damage.
A
oxygen (primary process) and electron transfer causing decar-
a
Dipartimento di Scienze Chimiche, Università di Catania, Viale Andrea Doria 6,
2
5
boxylation of the drug (secondary process). Indeed, NAP
photodegradation leads to a major photoproduct, 6-methoxy-2-
acetonaphthone (6-MAN), via involvement of reactive oxygen
9
5125 Catania, Italy. E-mail: gdeguidi@unict.it
b
Inca – Consorzio Interuniversitario Chimica per l’Ambiente, Via delle Industrie
2
1/8, 30175 Marghera, VE, Italy
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species (ROS) and with probable formation of a peroxide inter- Cell culture
mediate. The photoionization with free radicals production
leads to the decarboxylation process.
Yeast Saccharomyces cerevisiae wild type strain V328 (Chimica
Franke, Susa, TO, Italy) was grown in 50 mL of YPD broth (50 g L )
at about 30 °C to OD600 nm = 1.0. All subsequent treatments
were made in the dark.
2
5
−1
In two of our previous papers, an array of known Trp
photo) products used as markers for mechanism charac-
(
terization allowed us to shed more light on the drug photo-
2
6
induced damage on this amino acid. Trp was both free and Drug treatment
2
7
derived from bovine and human serum albumin (BSA/HSA).
Cells were harvested by centrifugation (3000g, 5 min), washed
In these works, MB and NAP induced Trp photodegradation
led to formation of several products such as kynurenine,
xanthurenic acid, N-formylkynurenine, 3-OH-kynurenine, 5-OH-
tryptophan, kynurenic acid, 4-OH-chinoline, and tryptamine.
On the other hand, the formation of alanine, serine and
aspartic acid (as Trp photodegradation products) was not
observed. The patterns of Trp photoproducts formed by the
two drugs are different and are specific for each type (I/II)
of UVA mediated sensitization process. These papers rep-
resented a key starting point for analysis of drug photosensiti-
zation on Trp of yeast Saccharomyces cerevisiae cells. The
choice of the more complex yeast model for MB or NAP photo-
sensitization on Trp is justified by low cost, ready availability
and mainly by its importance as a general cell model for
several metabolic processes. In addition, human cells and
yeast have many similar proteins for amino acid composition
and/or function. The results obtained from the cell model are
compared with data previously obtained from the simpler
with PBS and resuspended in a 200 mL quartz vessel with
900 μM of NAP or 100 μM of MB in PBS solution. Cells were
then incubated for 1 h in the dark at room temperature,
washed twice with cold PBS, resuspended in cold PBS (1.8 ×
6
−1
10
cells mL ) and exposed to increasing doses of UVA
radiation.
Irradiation condition
Cells were irradiated at 4 °C (in order to prevent metabolism) in a
−2
Rayonet photochemical reactor at a fluence rate of 1.0 mW cm
with eight “black light” phosphorous lamps (RPR-3500,
Southern N.E. Ultraviolet Co.) emitting virtually exclusively in
the UVA range with a peak at 350 nm. A stirrer located at the
bottom of the vessel continuously agitated the cell suspension.
The incident photon flux on the irradiated solution in the
vessel (200 mL), which is of the same order as the solar flu-
16
−1
ence incident on skin, was in the range of 1 × 10 quanta s
.
2
6,27
Immediately after irradiation, 1 mL of the cell suspension
was removed from the sample for the cell viability test.
models.
Cell viability
For all experiments, untreated and treated cells were diluted
and plated on solid YPD agar (65 g L− ). Colony forming units
1
(
CFU) were scored after 3 days of growth at about 30 °C. All
experiments were carried out independently at least 3 times.
Alkaline and enzymatic protein hydrolysis
Immediately after irradiation, the solvent was removed from
the samples by centrifugation (3000g, 5 min) and the cell
pellet hydrolyzed with 1000 U lyticase for 2 hours at room
temperature. 3 mL lysis buffer were added and incubated for
1 hour at 37 °C. In order to precipitate proteins, 12 mL ice cold
acetone were added to the sample and incubated overnight at
Materials and methods
Materials
NAP (naproxen), Trp (tryptophan), MB (methylene blue),
kynurenine, xanthurenic acid, 3-OH-kynurenine, 5-OH-trypto-
phan, kynurenic acid, 4-OH-chinoline, tryptamine, NaOH,
HCl, lyticase from Arthrobacter luteus, carboxypeptidase A from
bovine pancreas, protease type XIV from Streptomyces griseus
−20 °C. The sample was centrifuged at 10 000g for 20 min and
the protein pellet sample stored in a freezer. Alkaline and enzy-
matic protein hydrolysis, as well as evaluation of hydrolysis
27
reliability, were carried out according to the literature.
(
pronase E), YPD medium and YPD agar were obtained
Characterization of sensitizers, target and their photoproducts
from Sigma-Aldrich (Milano, Italy). N-Formylkynurenine was
synthesized as described previously.
2
6
Methanol (MeOH), Sensitizers, the target and their photoproducts were monitored
acetonitrile (ACN) and pentanesulfonic acid were of HPLC by HPLC/DAD/FLD/MSD analysis using three different analyti-
grade. All other chemicals were of reagent grade. PB cal methods named Systems 1, 2 and 3. Details are summar-
2
6,27
(
phosphate buffer) consists of sodium phosphate 10 mM at ized in Table 1; the literature reports major details.
pH 7.4; PBS (phosphate saline buffer) was made with sodium
phosphate 10 mM at pH 7.4, NaCl 138 mM and KCl 2.7 mM.
Calculation of quantum yields
All preparations were made by using Millipore bidistilled For the black light phosphor lamps, the light intensity measure-
2
6
water.
ments have been described previously.
The quantum
9
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a
Table 1 HPLC analysis systems
System 1
System 2
Formate/MeOH (gradient)
Detector
SOS/MeOH (isocratic)
Compound
t
R
t
R
Detector
DAD280
MB
NAP
Trp
7.9
21
8.1
10
9.5
3.8
5.5
5.8
DAD330–FLD330/360
DAD278–FLD278/356
DAD280–FLD278/356
DAD280
DAD360–FLD372/425
DAD360–FLD360/480
DAD330–FLD325/435
5.5
10
9.5
3.8
5.5
5.8
DAD278–FLD278/356
DAD280–FLD278/356
DAD280–FLD278/356
DAD360–ECD
DAD360–ECD
DAD330–ECD
4
-OH-chinoline
Tryptamine
-OH-kynurenine
3
Kynurenine
N-Formylkynurenine
Xanthurenic acid
Kynurenic acid
ECD
8.3
7.2
DAD330–FLD330/375
DAD280–FLD278/356
8.3
7.2
DAD330–FLD330/375
DAD280–FLD278/356
5-OH-tryptophan
a
26
System 3, used for determination of MB, consists of MSD flow injection analysis (FIA) in MeOH.
yield Φ of the investigated processes (sensitizer and target concentration of 90 μM. Non-irradiated cells incubated with
photodegradation, as well as their photoproducts formation, 900 μM NAP gave an intracellular drug concentration of
generally indicated as X) can be calculated from the initial rate 130 μM.
of each process, which equals:
Intracellular drug photodegradation was monitored for up
to 10% transformation for comparison with target induced
photodamage and evaluation of the quantum yields (see
below).
d½Xꢁ ϕFI
ꢀ
¼
v
dt
F = 1 − 10− is the fraction of light absorbed by the sensi-
tizer (Ā represents the mean absorbance of the sensitizer over-
lapping the emission range of the black light lamps), I is the
Ā
Drug photomediated Trp degradation
Cellular Trp is present in a free amino acid form and as bound
to proteins. 1 g of this amino acid is present in 100 g yeast pro-
−
1
measured light intensity (mol of photons min ) and v is the
volume of the irradiated sample. The calculation of quantum
yield takes into account the percentage of degradation that
occurs during alkaline hydrolysis of the protein.
teins and each cell has a total of 6 × 10− g proteins.
12
28,29
−
14
−1
Thus, it results in a Trp cell content of 6 × 10
g cell (2.94
−
16
−1
×
10
mol cell ). Therefore, each 200 mL non-irradiated
8
sample, containing 3.6 × 10 cells should have 21.6 μg Trp,
which is about 0.1 μmol Trp in the cells. The literature also
reports small amounts of Trp photoproducts, some of which
Analytical instrumentation
The HPLC analyses of samples were performed using a
Hewlett-Packard 1100 chromatograph equipped with an on-
line diode array detector (DAD), a Kontron SFM fluorescence
detector (FLD) and an ESA Coulochem electrochemical detec-
tor Mod 5100 (EC) (ESA, Inc., Bedford, MA) with a 5011 high
sensitivity analytical cell (porous graphite) and a model
2
6
are also metabolites; they should be taken into account for
2
,3
the quantitative determination of cell damage.
Fig. 1 shows MB photomediated Trp degradation and the
formation of Trp photoproducts. After 20 min of irradiation,
when about 20% of Trp is degraded, the Trp derivatives N-for-
mylkynurenine, kynurenine, tryptamine and 4-OH-chinoline
reach the maximum concentrations. N-Formylkynurenine and
5
020 guard cell, run in the screen mode. Mass spectra were
recorded with an Agilent VL mass spectrometer (MSD) sup-
plied with an API-ES interface.
3
0,31
kynurenine are type II diagnostic photoproducts,
whereas
tryptamine is formed through a type I mechanism and 4-OH-
3
2
chinoline is a mixed type I/II photoproduct. These results
confirm that MB acts mainly by a singlet oxygen photosensiti-
zation mechanism. Indeed, MB is a well known singlet oxygen
Results and discussion
Intracellular sensitizer photodegradation
9
,11,14,16,33,34
photosensitizer.
The resulting damage of samples
irradiated in the absence of drugs (controls) was negligible
For intracellular drug measurements, the samples were treated (data not shown).
as for Trp photoproduct quantitative determination, with the
Fig. 2 shows NAP photomediated Trp degradation and the
exception of protein hydrolysis. Thus, non-irradiated cell formation of Trp photoproducts. It is evident that after 20 min
samples, treated with 100 μM MB (200 mL), were centrifuged, of irradiation more than 30% of Trp is degraded; a higher
hydrolyzed, treated with lysis buffer and analyzed with HPLC. value with respect to MB photosensitization. Within this
Taking into account a single cell volume of 120 fL, the non- irradiation time, almost all Trp derivatives reach their
irradiated MB treated cells gave an intracellular drug maximum concentrations. Different to what happens in MB
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photosensitization, tryptamine (type I) and 4-OH-chinoline
Quantum yield calculations of amino acid degradation
(
type I/II) are formed in higher concentrations. These results (ΦTrp), as well as photoproducts formation, allow us to make
seem to confirm that NAP acts also by a radical photosensitiza- an accurate rationale (Table 2). ΦTrp are 9.1 × 10− and 2.5 ×
4
−
3
tion mechanism.
10
for MB and for NAP photosensitization, respectively.
Moreover, the quantum yields of photoproducts formation
confirm essentially the results obtained in simpler
2
6,35
models.
Indeed, in the presence of MB, the quantum yield
of the type II diagnostic N-formylated photoproduct is higher
than that in NAP photosensitization, whereas NAP photoin-
duces generation of the mixed type I/II product 4-OH-chinoline
−4
with quantum yield (3.5 × 10 ) higher than MB photosensiti-
zation (4.5 × 10− ). In addition, as expected, we observe a
decreased trend in the quantum efficiencies upon increasing
the model complexity; this behavior is evidenced in Fig. 3,
where a comparison of the quantum yields in free Trp, bound
to BSA, and from cells is presented. This fact can be explained,
considering the presence of a greater and greater number of
potential photosensitization targets. A parallel analysis of the
photodegradation quantum yields of the sensitizers gives
additional information for comparison among the three
5
Fig. 1 Photodegradation in cells of Trp (-■-) induced by MB and formation of models, and helps to correlate better with the quantum yields
their photoproducts: kynurenine (-●-), N-formylkynurenine (-▼-), tryptamine obtained for the target degradation and the photoproducts
(
-◆-) e 4-OH-chinoline (-▲-). Error bars represent a confidence interval of 5%.
formed. In Table 2 the values of the photodegradation
quantum yields calculated for MB and for NAP in the presence
of the different models are shown. The photodegradation
−
2
quantum yields of NAP and MB decrease in cells (1.8 × 10
and 0.1 × 10− ) if compared with those in the presence of free
2
Trp and BSA (6.4 and 6 × 10− NAP; 0.52 × 10 and 0.48 × 10
2
−2
−2
MB). The decrease of the quantum yield can be explained by
the fact that in the presence of free Trp and BSA, the unstable
transient species, generated under drug irradiation, are more
likely to be involved in drug photodegradation. This can be
due to the lower probability of the reaction of these transients
with a biomolecule, unlike what can happen in the intracel-
lular environment.
Cell viability
After 20 min irradiation, MB cell photosensitization (Fig. 4)
●
-), N-formylkynurenine (-▼-), tryptamine reduces survival of about 50% vs. a ΦTrp of 9.1 × 10 . At the
Fig. 2 Photodegradation in cells of Trp (-■-) induced by NAP and formation of
−
4
their photoproducts: kynurenine (-
-◆-) e 4-OH-chinoline (-▲-). Error bars represent a confidence interval of 5%.
(
same irradiation time, phototoxicity of NAP reduces the
2
a
Table 2 Quantum yields of the sensitizer and Trp photodegradation and Trp derivatives formation in simpler models and in yeast cells (values are expressed ×10 )
2
6
35
Free Trp
BSA
Yeast
MB
MB
NAP
MB
NAP
NAP
MB
NAP
Trp
0.52
0.48
0.1
6.4
1.0
6.0
0.8
1.8
0.25
0.62
0.625
0.091
4-OH-chinoline
0.15
0.067
0.2
0.07
0.32
0.006
0.81
0.45
0.4
0.02
0.09
0.016
0.15
0.18
0.0045
0.0065
nd
0.027
0.036
nd
0.035
0.025
nd
0.04
0.025
nd
Tryptamine
0.0075
0.051
0.02
0.08
0.0015
0.014
0.016
0.004
0.04
3-OH-kynurenine
Kynurenine
N-Formylkynurenine
Xanthurenic acid
0.0013
a
nd: detection below a value affordable for the calculation of quantum yields.
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photodegradation and higher Φ
Δ
could make MB a better type
II sensitizer for nuclear targeting and, consequently, for cell
viability.
Conclusions
The comparison of photosensitization quantum yields of MB
and NAP Trp photodamage in yeast represents a useful tool for
mechanistic studies of their photochemistry. This is particu-
larly true in very complex models like cells. In simpler models
the reaction mechanism is relatively easy to analyze, because
environmental conditions are controlled and most of the
unwanted interactions excluded.
The results obtained in cell NAP photosensitization are sup-
ported from those reported in the study of simpler models
Fig. 3 A comparison between the Trp photoproduct distributions in the three (free Trp, BSA and HSA); the mechanism is a mixed type I/II.
models, free Trp (26), BSA (27) and cells. Error bars represent a confidence inter-
In fact the diagnostic type I products are obtained in higher
yields with respect to the type II ones. Consequently, the NAP
val of 5%.
photosensitization mechanism on yeast Trp is a cooperative
type I/II, whereas MB photomediated cell amino acid damage
is mainly due to a type II mechanism. Also in this system,
results confirm the hypothesized mechanism proposed in free
Trp and in the isolated protein. Surprisingly, the overall cell
damage (cell viability) photomediated by MB is markedly
higher. This fact can be explained, considering the numerous
singlet oxygen targets in yeasts.
Therefore, we must pay serious attention to the evaluation
of the potential of the three proposed models. We do not want
to forget that sensitizers, once entered in the cell, can trigger
types of reactions that are influenced by several factors,
such as:
•
•
Modes of light absorption in a scattering system.
Concentration of the photosensitizer.
Fig. 4 Cell viability as measured by CFU (colony forming units) of MB (-●-)
and NAP (-■-) photosensitized yeast. Error bars represent a confidence interval
of 5%.
• Time of contact between the sensitizer and the cell.
• Transport of the sensitizer to the cell, and uptake in the
cell.
•
Sub-cellular localization of the sensitizer.
Moreover, an “in vitro” simulation must take into account
survival of about 30%. Considering the higher NAP photo- that photosensitizers can modify the outside of the cell and
−
3
mediated ΦTrp of 2.5 × 10 , we observe that amino acid photo- the extracellular matrix, probably by cross-linking and photo-
degradation and cell damage are not in strict agreement. This is oxidation of proteins. Again, the relative importance of
most likely due to the damage on other cell targets. Indeed, different types of damage (such as discrepancy between the
3
6,37
MB is able to photoinduce DNA damage by singlet oxygen,
efficiencies of photoinduced Trp oxidation and cell viability) is
chiefly influenced by the distance between the bound sensi-
3
3
34
promoting base oxidation both in free and genomic DNA.
Differently, NAP photomediated DNA damage ability is not so tizer, and the site where the damage can take place.
3
8,39
marked. This drug has been tested on free DNA
and
In conclusion, all three systems are equally feasible, since
3
5
human fibroblasts. NAP photocleaving activity on plasmid they do not differ dramatically in the wanted mechanistic
4
0
DNA resulted in induction of single strand breaks. In the photosensitized response.
presence of oxygen, the efficiency of NAP is increased. Litera-
4
0
33
ture data suggest that NAP and MB interact with DNA by
the same mechanism, i.e. type II. Consequently, the mechan-
ism of photoinduced DNA cleavage could probably strongly be
Abbreviations
influenced both by sensitizer photodegradation quantum yield NAP Naproxen
NAP 1.8 × 10− vs. MB 0.1 × 10 ) and singlet oxygen quantum MB Methylene blue
2
−2
(
2
4
10
yield (NAP 0.3
vs. MB 0.6 ). Concomitance of lower Trp Tryptophan
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partially inactivated enzyme, J. Photochem. Photobiol., B,
Acknowledgements
2000, 55, 196–200.
The authors thank INCA
– Consorzio Interuniversitario 14 A. Michaeli and J. Feitelson, Reactivity of singlet oxygen
Chimica per l’Ambiente (Marghera, VE, Italy) for providing
analytical instrumentation.
toward proteins: the effect of structure in basic pancreatic
trypsin inhibitor and in ribonuclease A, Photochem. Photo-
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1
5 S. G. Bertolotti, N. A. Garcia and G. A. Argüello, Effect
of the peptide bond on the singlet molecular
oxygen-mediated photo-oxidation of tyrosine and trypto-
phan dipeptides, J. Photochem. Photobiol., B, 1991, 10,
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