Current microbiology

fotoinativacao por derivados xantenos

1. The Phototoxicity of Xanthene Derivatives AgainstEscherichia coli,
Staphylococcus aureus, andSaccharomyces cerevisiae
Hong Wang, Lei Lu, Shiyun Zhu, Yahong Li, Weimin Cai
Schoolof EnvironmentalScience and Engineering, Shanghai Jiao Tong University, Shanghai 200240, China
Received: 8 March 2005 / Accepted: 11 September 2005
Abstract.We assessed the phototoxicity of a series of xanthene derivatives againstE.coli,S.aureus,
andS.cerevisiae, measured the physicochemicalproperties of the photosensitizers, and found the
relationship between them. Without illumination, the dyes tested showed almost the same level of
inherent toxicity to the same organism, which showed the inherent toxicity of dyes was primarily
dependent on the structure of parent molecule. Upon illumination, the photosensitizers showed obvious
phototoxicity to all organisms. The dyes showed stronger phototoxicity to Gram-positive bacteria. With
the increasing number of halogen substituents, the singlet oxygen yields increased and the phototoxic
activity increased too. There was no obvious correlation between relative lipophilicity and activity in the
current study. Our results showed xanthenes had the potential to act as alternatives to conventional
antimicrobial compounds and also could be used for the decontamination of microbially polluted waters.
The research field of antimicrobial compounds is one
of the current constant challenges. Unfortunately, an
unpleasant cycle has recently appeared: as soon as a new
drug is introduced, the strains resistant to that drug
emerge [14]. There is an urgent need for some new
drugs with novelmechanisms of attack.
Extensive research had been carried out to screen
the photosensitizers with antimicrobialproperties and
illuminate their photodynamic mechanisms [2, 3, 19,
20]. Photosensitizers can be electronically excited by
irradiation with light at the wavelength appropriate for
that photosensitizer after being localized in the target
organism [4]. Then the molecule will pass its excitation
energy onto other biomolecules by two mechanisms. In
the type I process, energy can be transferred between the
excited photosensitizer and nearby biomolecules,
yielding oxygenated free radicals; In the type II process,
energy can be transferred between the excited photo-
sensitizer and molecular oxygen, yielding singlet oxy-
gen,
1
O2[6]. Such highly reactive products are able to
photomodify some biomolecules in cells such as lipids,
enzymes, and DNA [7, 11, 12], which will be lethal to
cells. The non-specific nature of these attacking modes
makes it unlikely for the bacteria to acquire the resis-
tance to the photosensitizers.
Many photosensitizers had been shown to possess
antimicrobialproperties [20]. Xanthene derivatives were
a class of photosensitizing molecules and some of them
had been developed for commercial use as pesticides [5,
9]. In the present study, the phototoxicity of a series
of xanthene derivatives againstE.coli,S.aureus, and
S.cerevisiae, was assessed.
Materials and Methods
Photosensitizers.All chemicals used in our experiments were of
analytical grade. Fluorescein (Fl)-derived photosensitizers (Table 1)
were purchased from Shanghai ChemicalRegents Company (China).
Na
2Fl, Na2FlBr4,Na2FlI4, and Na2FlBr4Cl4were stored as aqueous
stock solutions (1 m
M)at4TC. FlBr
2and FlI
2were stored as 95%
ethanolstock solutions (1 m
M)at4TC.
Light source.150-W metal halide lamp (Philips, The Netherlands),
giving a light fluence of 15 mW cm
)2
, was used in toxicity tests. The
fluence of the polychromatic light was measured with a FZ-A light
meter (Handy, China).
Physicochemical properities.The lipophilicities of the photosen-
sitizers were calculated in terms of logP, the logarithm of their
partition coefficients between 1-octanoland deionized water. MaximalCorrespondence to:Hong Wang;email:[email protected]
C
URRENTMICROBIOLOGYVol. 52 (2006), pp. 1–5
DOI: 10.1007/s00284-005-0040-z
Current
Microbiology
An International Journal
ªSpringer Science+Business Media, Inc. 2005

2. dye absorption in the range of visible light was determined in aqueous
solutions by a Unico 2102 UV/Vis spectrophotometer (Japan). The
photosensitizing efficiency was measured as relative singlet oxygen
yield (F¢). According to the method described by Kraljic and Mohsni
[13], the system imidazole plusp-nitrosodimethylaniline (RNO) can be
used as a sensitive and selective test for the presence of
1
O
2in aqueous
solutions. The test is based on secondary bleaching of RNO as induced by
the reaction of
1
O2with imidazole. In the present study, the solutions of
different photosensitizers were irradiated in closed absorption cells of
10-mm light path for 5 min and the O.D. was determined before and after
irradiation at 440 nm, the maximalabsorption of RNO. The
concentrations of photosensitizer, RNO, and imidazole were set to
30l
M,50l Mand 8 mM, respectively. The experiments were carried out
at room temperature (25TC). We setF¢=1 for Na
2FlBr
4Cl
4.
Bacterial strains and growth condition.E.coli(AS 1.129) andS.
aureus(AS 1.72) were grown aerobically in liquid LB medium for
approximately 18 h using an orbital incubator at 37TC, which
corresponded to the cell concentration of around 2·10
9
cfu ml
)1
.
S.cerevisiae(AS 2.101) was grown in liquid PDA medium at 28TC for
approximately 24 h, which corresponded to around 2·10
8
cfu ml
)1
.
For the phototoxicity tests, cells were diluted with fresh medium to a
finalcellconcentration of 10
5
cfu ml
)1
.
Toxicity tests.Qualitative phototoxicity tests were carried out using
the methods modified from Daniels [1]. Photosensitizers were
dissolved in corresponding solvents to the concentration of 2 mg/
mL. Five microliters of each solution was applied to a piece of filter
paper disc (7-mm diameter, 10lg/disc) and solvents were allowed to
evaporate in the dark. Forty microliters of cells at the concentration of
10
5
cfu ml
)1
was spread onto a plate evenly with a sterile bent glass
rod. The discs were placed onto the freshly inoculated plates and all
plates were incubated in dark for 30 min. Then, after 30-min irradiation
period under the light fluence of 15 mW cm
)2
, all plates were
incubated in dark again at the temperature mentioned above for another
48 h. The zones of inhibition were measured. For the dark control
plates, we omitted the 30-min irradiation period. All assays were
repeated 3 times and the results combined.
Toxicity tests were conducted as 10-fold replicates using 300-lL
flat-bottomed 96-well micro-titre plates (Costar). Fresh cell suspen-
sions were prepared as described above. These suspensions were then
mixed with the chemicals shown in Table 1 to obtain twofold dilution
series of each photosensitizer with the finalconcentrations varying
from 1 to 500l
M. Two hundred microliters of these mixtures were
then added to the wells of micro-titre plates and all plates were incu-
bated in dark for 30 min. Then, after a 30-min irradiation period under
the light fluence of 15 mW cm
)2
, all plates were placed in an incubator
again in dark at the temperature mentioned above for 18 h. The
resulting 10-lL cultures were streaked onto agar medium plates and
incubated for another 18 h. After incubation, tested plates were
examined for bacterialgrowth and the lowest concentration at which
no colonies were observed was taken as the minimum lethal concen-
tration (MLC) of a given photosensitizer. The experiments were re-
peated untilthe same results appeared.
Results and Discussion
The MLCs of Na
2Fl, FlBr
2,FlI
2,Na
2FlBr
4,Na
2FlI
4, and
Na
2FlBr
4Cl
4(structures shown in Table 1) were deter-
mined when directly against three microorganisms with
or without illumination (Table 2). The results were
consistent with the qualitative phototoxicity assays
(Table 3). The spectral output of the light source coin-
Table 1. Structures of photosensitizers
Photosensitizer XYZ
Fluorescein, disodium salt (Na
2Fl) HHH
Dibromofluorescein (FlBr
2) Br H H
Diiodofluorescein (FlI
2) IHH
Tetrabromofluorescein. disodium salt (Na
2FlBr4) BrBrH
Tetraiodofluorecein. disodium salt (Na
2FlI4) IIH
Tetrachlorotetrabromofluorescein. disodium salt (Na
2FlBr
4Cl
4) BrBrCl
2 CURRENTMICROBIOLOGYVol. 52 (2006)

3. cided with the max absorption wavelength of the
photosensitizers. Controlexperiments showed that in the
absence of photosensitizers, illumination alone or etha-
nol-added medium had no effect on all organisms (data
not shown).
Without illumination, Na
2Flshowed no inherent
toxicity to three organisms under the concentration as
high as 500l
M. The inherent MLCs of the other
photosensitizers toE.coli,S.aureus, andS.cerevisiae
were 500l
M, 62.5 to 125l M, and 125 to 250l M,
respectively. In the present study, all photosensitizers
showed almost the same level of inherent toxicity to the
same organism. The present study showed that the
inherent toxicity was primarily dependent on the struc-
ture of parent molecule, and substituted groups had
slight effects on the inherent activity.
Upon illumination, the photosensitizers showed
obvious phototoxicity against either bacterium under
the conditions in our experiments. The xanthenes
showed stronger phototoxicity to Gram-positive bacte-
ria.S. cerevisiaewas found to be much more sensitive
than bacteria to the same xanthene. Na2FlI
4and
Na
2FlBr4Cl4were the most phototoxic dyes tested with
the MLCs of 125, 1, and 1l
Mwhen directly against
E.coli,S.aureus, andS.cerevisiae, respectively. The
physicochemicalproperities of the photosensitizers
used in the present study are given in Table 4. In vitro
chemicaltests showed that each of the xanthene
derivatives was able to photosensitize the production of
singlet oxygen, in the order of Na
2FlBr4Cl4>Na2FlI4
>Na2FlBr4>FlI2>FlBr2>Na2Fl. All dyes tested
except Na
2Flwere phototoxic to three organisms and
generated significant levels of singlet oxygen. It is
suggested that type II mechanism of photosensitization
played an important role in such actions. With the
increasing number of halogen substituents, the singlet
oxygen yields increased and the phototoxic activity
increased too. Some research has shown that the
Table 2. Toxicity of photosensitizers againstE. coil,S. aureus, andS. cerevisiae
MLC (l
M)
E. coli S. aureus S. cerevisiae
Photosensitizers Dark Light Dark/Light Dark Light Dark/Light Dark Light Dark/Light
Na
2Fl )) ) ) ) 15.6
FlBr
2 500 250 2 125 31.3 4 250 3.9 64
FlI
2 500 125 4 125 31.3 4 250 3.9 64
Na
2FlBr4 500 125 4 125 2 62.5 250 2 125
Na
2FlI
4 500 125 4 62.5 1 62.5 250 1 250
Na
2FlBr4Cl4 500 125 4 62.5 1 62.5 125 1 125
)= no obvious effects under the highest concentration.
Table 3. Qualitative phototoxicity assays
E. coli S. aureus S. cerevisiae
Photosensitizers Dark Light Dark Light Dark Light
Na
2Fl ))))) +
FlBr
2 ) +++ ) ++
FlI
2 ) +++ ) ++
Na
2FlBr4 ) ++++ ) ++
Na
2FlI
4 ) ++++ ) ++
Na
2FlBr4Cl4 ) ++++ ) ++
Clear zone diameter + = 1–5 cm; ++ = 5–10 cm;)= no obvious effects.
Table 4. Physicochemical properties of photosensitizers
Photosensitizer k
max(nm)
a
LogP F¢
b
Na
2Fl490 )0.28 0.02
FlBr
2 504 1.01 0.67
FlI
2 507 1.21 0.69
Na
2FlBr
4 517 )0.25 0.81
Na
2FlI
4 527 )0.24 0.92
Na
2FlBr4Cl4 539 )0.21 1
a
Measured in water.
b
Relative to the singlet oxygen yield of Na
2FlBr
4Cl
4.
H. Wang et al.: The Phototoxicity of Xanthene Derivatives
3

4. presence of heavy bromine or iodine atoms enhanced
the yields of intersystem crossing to the reactive triplet
state of the xanthene dyes [11].
The LogPvalues measured in the experiments
showed that FlBr
2and FlI2were lipophilic (LogP>
0), while Na
2Fl, Na
2FlBr
4,Na
2FlI
4, and Na
2FlBr
4Cl
4
were hydrophilic (LogP< 0). Variation in LogPwas
expected to affect the uptake and localization of the
photosensitizers. In the current study, there was no
obvious correlation between relative lipophilicity and
activity. Due to the work of Pooler and Valenzeno
[18], we knew that xanthenes were typically localized
in cell membrane. At a molecular level, xanthenes
mostly photosensitized the cross-linking of proteins
and formed the hydroperoxides from unsaturated lip-
ids, thereby increasing the osmotic fragility of the
cells. It was not necessary for such photosensitizers to
pass through the membrane to attack intracellular
targets. However, Pimprikar and Heitz [17] observed
the toxicity ratio toAedesmosquito larvae ranged up
to 2 orders of magnitude between the soluble and
insoluble forms of the same xanthene dye. With the
lipophilic xanthenes, mosquito larvae were able to
filter feed on dye particles and thereby received a
higher level of the dye. Thus, lipophilic xanthenes
showed higher activity against mosquito larvae than
organisms in present tests.
There was a clear tendency that xanthenes showed
higher activity against Gram-positive bacteria. The
additionallayer of protection provided by the outer
membrane of Gram-negative bacteria could generally
hinder the binding of the photosensitizers and intercept
photo-generated reactive species. Researchers drew the
same conclusion in a previous study [16].
In the present study, photosensitizers showed rela-
tively higher toxicity upon illumination and lower tox-
icity without illumination against yeastS.cerevisiae
than bacteria. Yeast was shown to be more sensitive
to phototoxic reaction. Thus, it may be considered that
S. cerevisiaeis better thanE. coliorS. aureusin
screening usefulphotosensitizers.
Our results showed the dyes tested here could kill
bacteria and yeast at micromolar concentrations. In
relation to therapeutic use, although several photo-
sensitizers have been used successfully in photother-
apy, the dyes tested here still need some possible
structuralmodifications to make theirk
maxlie within
the window of 600–900 nm used for the treatment of
human conditions. All the tested dyes will not persist
or remain toxic for a very long time in the environ-
ment. Previous research showed that exposure of
Na
2Flor Na2FlBr4Cl4to sunlight was expected to lead
to photodegradation with a half time of approximately
1 hour [10]. Thus, Suredye (69% Na
2FlBr4Cl4and
31% Na
2Flby weight of active components) becomes
the most successfully used photoinsecticide to control
the fruit fly [8]. In addition, Na
2FlBr4Cl4had been
approved by EPA (USA) for trialapplications to
controlcorn root worms in 2000 and some more of
these dyes were in experiments for insect control. At
the same time, some researchers stated the photosen-
sitized inactivation of yeast and bacteria could be used
for the decontamination of microbially polluted waters
[15]. Thus, photoactivated dyes show potentialas
antimicrobialagents; additionalstudies are needed to
define and explore their applications for biological
controland decontamination.
ACKNOWLEDGMENTS
We thank Dr. Xiaofan Zhang for his technicalassistance.
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