Photoinduced nitric oxide release from a nitrobenzene derivative in mitochondria

Article abstract of DOI:10.1002/chem.201001967

We report a novel NO donor (RpNO), containing a 2,6-dimethylnitrobenzene moiety for photocontrollable NO release and a rhodamine moiety for targeting to mitochondria. Photorelease of NO from RpNO in aqueous solution was confirmed by means of ESR analysis. Cellular release of NO from RpNO was confirmed with the aid of DAF-FMDA, an NO-specific fluorescence probe. RpNO was colocalized with MitoTracker GreenFM, a mitochondrial stain, in HCT116 colon cancer cells and exhibited photodependent cytotoxicity. Our results indicate that RpNO is an effective NO donor for time-controlled, mitochondria-specific NO treatment. NO entry! A novel NO donor (RpNO) containing a 2,6-dimethylnitrobenzene moiety for photocontrollable NO release and a rhodamine moiety for targeting to mitochondria was synthesized (see graphic). It was confirmed that RpNO became localized in the mitochondria of HCT116 colon cancer cells, and showed photodependent cytotoxicity. This is the first example of a photocontrollable, mitochondria-localizing NO donor. Copyright

Full text of DOI:10.1002/chem.201001967

FULL PAPER
DOI: 10.1002/chem.201001967
Photoinduced Nitric Oxide Release from a Nitrobenzene Derivative in
Mitochondria
Taeko Horinouchi,[a] Hidehiko Nakagawa,*[a, c] Takayoshi Suzuki,[a]
Kiyoshi Fukuhara,[b] and Naoki Miyata*[a, b]
Abstract: We report a novel NO donor
(RpNO), containing a 2,6-dimethylni-
trobenzene moiety for photocontrolla-
of NO from RpNO was confirmed with
the aid of DAF-FM DA, an NO-specif-
ic fluorescence probe. RpNO was colo-
calized with MitoTracker Green FM, a
mitochondrial stain, in HCT116 colon
cancer cells and exhibited photode-
pendent cytotoxicity. Our results indi-
cate that RpNO is an effective NO
donor for time-controlled, mitochon-
dria-specific NO treatment.
ble NO release and
a rhodamine
moiety for targeting to mitochondria.
Photorelease of NO from RpNO in
aqueous solution was confirmed by
means of ESR analysis. Cellular release
Keywords: caged compounds · cy-
totoxicity · mitochondria · NO do-
nor · photolysis
Introduction
have also demonstrated that our 2,6-dimethylnitrobenzene
derivatives work as photocontrollable NO donors in
HCT116 human colon cancer cells. Because concentrations
and the biological influence of NO differ in different organ-
elles, organelle-specific photocontrollable NO donors would
be extremely useful.[9] Because the mitochondrion is a key
organelle for apoptotic signal transduction, and because NO
induces cellular apoptosis through mitochondrion-dependent
pathways, mitochondria are a potential target for NO
donors to initiate apoptotic cancer cell death.[10] Further-
more, after recent reports of mitochondrial nitric oxide syn-
thase activity,[11] mitochondria-specific NO donors would
also be useful for elucidation of NO functions in mitochon-
dria.
With the aim of developing functional photoactivatable
NO donors, we designed and synthesized RpNO (1), con-
taining both a 2,6-dimethylnitrobenzene moiety for photo-
controllable NO release and a rhodamine moiety for target-
ing to mitochondria. The rhodamine moiety effectively tar-
gets mitochondria as a result of its hydrophobic cationic
character.[12]
Nitric oxide (NO), although it exists as an unstable free rad-
ical under ambient conditions, is involved in the mainte-
nance and regulation of many vital functions,[1] serving as,
for example, a pleiotropic bioregulator of blood pressure, a
neuromodulator, and a biodefense agent. Consequently,
many NO donors have been developed and employed for
biological studies.[2] However, most NO donors currently
used, such as NOCs (secondary amine-derived diazenium di-
olates)[3] and NORs (a,b-unsaturated oxime derivatives),[4]
release NO by spontaneous autolysis. Photocontrollable NO
donors are far more attractive tools, allowing both spatial
and temporal control of NO release.[5] Although sodium ni-
troprusside (SNP) is available as a photoinducible NO
donor, it has the drawback of potential toxicity due to the
cyanide ligands.[6]
We have previously reported 4-substituted 2,6-dimethylni-
trobenzenes as a new type of NO donor.[7] Irradiation of
2,6-dimethylnitrobenzenes with extended p-electron systems
at their 4-positions with ultraviolet A light results in efficient
NO release as a result of rearrangement to arylnitrites. We
[a] T. Horinouchi, Dr. H. Nakagawa, Dr. T. Suzuki, Prof. Dr. N. Miyata
Graduate School of Pharmaceutical Sciences, Nagoya City University
3–1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603 (Japan)
Fax : (+81)52-836-3407
E-mail: deco@phar.nagoya-cu.ac.jp
miyata-n@phar.nagoya-cu.ac.jp
[b] Dr. K. Fukuhara, Prof. Dr. N. Miyata
Organic Chemistry, National Institute of Health Sciences
Setagaya, Tokyo 158-8501 (Japan)
[c] Dr. H. Nakagawa
Here we show that our novel NO donor RpNO is distrib-
uted exclusively to the mitochondria, where it releases NO
in response to UV-A irradiation. Its irradiation-dependent
cytotoxicity towards HCT116 cells was also evaluated.
PRESTO (Japan) Science and Technology Agency (JST)
4-1-8 Honcho Kawaguchi, Saitama 332-0012 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201001967.
Chem. Eur. J. 2011, 17, 4809 – 4813
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4809

Results and Discussion
RpNO was photoirradiated in MilliQ water containing
DMSO (7.5%) for 15 min in the presence of Fe-MGD, a
three-line spectrum typical of [(MGD)-Fe2+-NO] was ob-
served by ESR spectroscopy, indicating that NO had been
released from RpNO upon photoirradiation (Figure 1).
Molecular design and synthesis: We focused on use of the
rhodamine component to achieve mitochondrial localization
and of the 2,6-dimethylnitrobenzene unit as the NO-releas-
ing moiety, and aimed to couple commercially available rho-
damine B to the 2,6-dimethylnitrobenzene moiety at the
2’-carbonyl group (Scheme 1).[13] Formation of a tertiary
Figure 1. A representative ESR spectrum of a solution containing Fe-
MGD and RpNO after photoirradiation (330–380 nm) with a 6% ND fil-
ter. Samples contained RpNO (750 mm), MGD (60 mm), and FeSO4
(15 mm) in MilliQ water containing DMSO (7.5%). ESR spectra were re-
corded after photoirradiation for 15 min with a modulation width of
1.25 G and a microwave power of 10 mW.
From a calibration curve prepared with [(MGD)-Fe2+-NO],
the rate of NO release from RpNO was 6% upon 15 min
photoirradiation with the light source (100 W mercury
lamp) of a fluorescence microscope fitted with a WU filter
(330–380 nm band-pass filter) and a 6% ND filter (see Fig-
ure S4 in the Supporting Information).
Confocal microscopy: For confocal microscopy, HCT116
cells were cultured in McCoyꢁs 5 A culture medium. For the
experiments, cells were incubated for 2 days and then
washed with D-PBS (Dulbeccoꢁs PBS) and treated with
RpNO (10 nm) for 5 min in the dark, followed by washing
once with D-PBS. The cells were then stained for 15 min
with MitoTracker Green FM, a well-established mitochon-
drial dye, and subjected to confocal fluorescence microsco-
py. RpNO was found to be localized in the mitochondria
(Figure 2, and Figure S5 in the Supporting Information), as
would be expected in view of the presence of the mem-
brane-permeable and cationic rhodamine moiety, because of
Scheme 1. Synthesis of RpNO (1). Reagents and conditions; a) nitric acid
(70%), 1008C, 1 h, 49%; b) (Boc)2O, DMAP, tBuOH, RT, 3 h, 75%; c) 3,
PdACHTUNGTRENNUNG(OAc)2, PPh3, Et3N, AgNO3, MeCN, 1008C, 3.5 h, 67%; d) TFA,
CHCl3, RT, 93%; e) 7, (COCl)2, DMF, CH2Cl2, RT, 5 h, 45%. Boc=tert-
butoxycarbonyl, DMAP=4-dimethylaminopyridine.
amide bond with piperazine as a linker moiety prevents cyc-
lization of RpNO into a nonfluorescent lactam form.[14] The
NO donor moiety was thus connected to a conjugated
p-electron system known to be photoresponsive in biological
applications. RpNO was synthesized as shown in Scheme 1
1
and characterized by H and 13C NMR spectroscopy, HPLC,
melting point measurement, and mass spectroscopy.
Release of NO from RpNO in vitro: NO release from
RpNO was determined by an ESR spin-trapping method
with an Fe-N-methyl-d-glucamine dithiocarbamate (MGD)
complex, which is known to react with NO to give the stable
paramagnetic complex [(MGD)-Fe2+-NO]. In its ESR spec-
trum, the [(MGD)-Fe2+-NO] complex gives a broadened
three-line signal with aN =1.25 mT and giso =2.04. When
Figure 2. HCT116 human colon cancer cells were stained with MitoTrack-
er Green FM and RpNO and observed under a confocal fluorescence mi-
croscope. The distribution of MitoTracker Green FM (left, green), the
distribution of RpNO (center, red), and the merged image (right) of the
same field are shown.
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Photoinduced NO Release from a Nitrobenzene Derivative in Mitochondria
FULL PAPER
the highly inside-negative membrane potential across the
inner mitochondrial membrane.
Intracellular release of NO from RpNO: Photorelease of
NO from RpNO in HCT116 cells was confirmed with the
aid of DAF-FM DA,[15] a cell-membrane-permeable NO-spe-
cific fluorescence probe. DAF-FM DA itself is a nonfluores-
cent compound, but is known to be hydrolyzed and to react
with NO to form triazole DAF-FM T, which has strong
green fluorescence. For confocal microscopy, the cells were
plated and cultured for 2 days and were then treated with
RpNO (100 mm) in the same manner as in the colocalization
experiment. DAF-FM DA was loaded into the cells for
10 min and the cells then were irradiated with UV-A light
for 5 min. Confocal microscopic observation revealed that
the cells showed green fluorescence derived from DAF-
FM T only when exposed to both RpNO and UV light. This
suggested that RpNO released NO in the cells upon photoir-
Figure 4. Cytotoxicity of NO released from RpNO under photoirradia-
tion conditions. Representative phase-contrast images of cells within the
irradiation area A) in the absence of P450 nor, or B) in the presence of
P450 nor are shown.
radiation (Figure 3), or put another way, that RpNO acts as
a photocontrollable NO donor (see also Figure S6 in the
Supporting Information).
dependent cytotoxicity was effectively diminished in the
presence of b-NADH and P450 nor,[17] an NO reductase
(Figure 4B). This indicates that the cytotoxicity is due to
NO release from RpNO under photoirradiation conditions
(see also Figure S7 in the Supporting Information).
Figure 3. Fluorescence images of HCT116 human colon cancer cells
loaded with RpNO and DAF-FM DA, left) before, and right) after pho-
toirradiation (330–380 nm) with a 1.5% ND filter. Samples contained
RpNO (100 mm) and DAF-FM DA (10 mm).
Conclusion
We have demonstrated photoinduced NO generation in the
mitochondria of living cells through the use of RpNO, a
2,6-dimethylnitrobenzene derivative linked to a rhodamine
dye, which serves to drive localization of RpNO into mito-
chondria. This is the first example of a photocontrollable
mitochondria-localizing NO donor. RpNO is expected be a
useful tool for studies of the biological roles of NO.
As shown in Figure 3, the cells were consistently stained
with green fluorescence, even though it had been confirmed
that RpNO was localized specifically in the mitochondria.
This is probably because the diffusion rates of DAF-FM T
or of NO itself are very fast, so that it is hard to identify the
location of NO production in this experiment, even though
RpNO is localized in mitochondria and so should release
NO there.
Experimental Section
General methods: Melting points were determined with a Yanagimoto
micromelting point apparatus. 1H and 13C NMR spectra were recorded
with a JEOL JNM-LA500 or a JEOL JNM-A500 spectrometer in the in-
dicated solvent. Chemical shifts (d) are reported in parts per million rela-
tive to tetramethylsilane as internal standard. Elemental analyses were
performed with a Yanaco CHN CORDER NT-5 analyzer, and all values
were within ꢀ0.4% of the calculated values. Fast atom bombardment
(FAB) mass spectra were recorded with a JEOL JMS-SX102 A mass
spectrometer. UV/Vis spectra were recorded with an Agilent 8453 spec-
trophotometer. MGD was obtained from Dojindo. All other reagents
and solvents were purchased from Aldrich, Tokyo Kasei Kogyo, Wako
Pure Chemical Industries, Nacalai Tesque, and Kanto Kagaku, and were
used without purification. Flash column chromatography was performed
with silica gel 60 (particle size 0.046–0.063 mm, Merck). Photoirradiation
was performed with use either of the light source of an Olympus BX-60
fluorescence microscope or of an Asahi Spectra MAX-302 irradiating ap-
paratus.
Cytotoxicity towards cancer cells: It has been reported that
NO mediates the cytotoxic action of macrophages towards
tumor cells through inhibition of mitochondrial enzyme ac-
tivity.[16] We had previously reported that nonlocalizing
2,6-dimethylnitrobenzene derivatives induced cytotoxicity in
HCT116 cells through the release of NO under photoirra-
diation conditions.[7] In the presence of RpNO (10 mm), light
in the wavelength range of 325–375 nm was applied for
5 min on a fixed area of a culture plate containing human
colon cancer HCT116 cells. The cells were observed after in-
cubation for 24 h at 378C in the dark. As expected, the cells
within the irradiated area were detached and appeared dead
(Figure 4A). Treatment either with RpNO alone or with
photoirradiation alone had no effect. This photoirradiation-
Chem. Eur. J. 2011, 17, 4809 – 4813
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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4811

H. Nakagawa, N. Miyata et al.
Synthesis
incubator (CO2, 5%, v/v) for 2 days. The dishes were then washed with
Dulbeccoꢁs PBS (D-PBS) and the medium was replaced with D-PBS
(1 mL). The cells were treated with RpNO (10 nm) for 5 min in the dark,
washed once with D-PBS, stained with MitoTracker Green FM (100 nm)
according to the manufacturerꢁs instructions, and subjected to confocal
fluorescence microscopy without washing.
Compound 3: 1-Iodo-3,5-dimethylbenzene (2, 8.04 g, 34.65 mmol) was
added at 08C to a solution of HNO3 (70%, 25 mL). The mixture was
stirred for 1 hour at 1008C and was then poured onto ice, neutralized
with saturated NaHCO3 solution, and extracted with Et2O. The Et2O
layer was washed with brine and dried over Na2SO4. Filtration and con-
centration afforded a residue, which was subjected to silica gel flash
Photoirradiation: For the ESR experiment, a quartz flat cuvette contain-
ing RpNO and a NO trap was irradiated by use of a conventional fluores-
cence microscope (Olympus BX-60, 100 W mercury lamp) with a WU
filter (330–380 nm band-pass filter, 3.84 mWcmꢁ2). For detection of NO
with DAF-FM DA, cells were irradiated with same microscope light
source as in the ESR experiment. For photoirradiation experiments, the
cells were placed at a distance of 10 cm from a lens attached to a MAX-
302 light source (Asahi Spectra, Tokyo) with a xenon lamp (300 W) and
irradiated with UV-A light (325–375 nm) through a 50% ND filter for
the time indicated (15.1 mWcmꢁ2).
ESR analysis: The Fe2+ complex of MGD (Fe-MGD) was used to trap
NO. A fresh solution of Fe-MGD (1:4) was prepared by addition of fer-
rous ammonium sulfate to a MilliQ water solution of MGD. A sample
containing RpNO (750 mm) and Fe-MGD (15 mm) in MilliQ water (7.5%
DMSO) was introduced into a quartz flat cuvette. Photoirradiation was
carried out for 15 min with the light source (100 W mercury lamp) of a
fluorescence microscope fitted with a WU filter (330–380 nm band-pass
filter) and a 6% ND filter, and ESR spectra were recorded with a JES-
RE 2X spectrometer (JEOL Co. Ltd., Tokyo, Japan). For the calibration
curve, standard solutions containing designated amounts of Fe-MGD
(12.5 mm to 400 mm) were treated with NOC7 (excess) in MilliQ water
(7.5% DMSO). The ESR signals of standard solutions were recorded
with an ESR spectrometer in the same manner, and the peak areas of
the NO complex were plotted against the concentration of [(MGD)-Fe2+
-NO] (see the Supporting Information). The spectrometer settings were
as follows: modulation frequency 100 kHz, modulation amplitude 2.0 G,
scan time 4 min, microwave power 16 mW, and microwave frequency
9.42 GHz.
column chromatography (toluene/n-hexane 1:20) to give
3 (4.60 g,
16.66 mmol, 48.1%) as a pale yellow solid: 1H NMR (CDCl3, 500 MHz):
d =7.51 (s, 2H), 2.27 ppm (s, 6H).
Compound 5: (Boc)2O (33.15 g, 152.08 mmol, 1.5 equiv) and DMAP
(24.78 g, 202.77 mmol, 2.0 equiv) were added to a solution of 4-ethenyl-
benzoic acid (4, 15.01 g, 101.39 mmol) in tert-butanol (500 mL). The mix-
ture was stirred for 3 h at room temperature and was then poured into
water and extracted with CHCl3. The CHCl3 layer was washed with brine
and dried over Na2SO4. Filtration and evaporation afforded a residue,
which was subjected to silica gel flash column chromatography (AcOEt/
n-hexane 1:10) to give 5 (15.68 g, 76.85 mmol, 75.8%) as a colorless oil:
1H NMR (CDCl3, 500 MHz):
J=8.2 Hz, 2H), 6.75 (dd, J=20.5 Hz, 11.0 Hz, 1H), 5.85 (d, J=17.7 Hz,
1H), 5.36 (d, J=10.7 Hz, 1H), 1.59 ppm (s, 9H).
d =7.94 (d, J=8.2 Hz, 2H), 7.44 (d,
Compound 6:
A
mixture of PdACHTNGUTERNNU(G OAc)2 (248.19 mg, 1.11 mmol,
0.10 equiv), PPh3 (608.36 mg, 2.32 mmol, 0.21 equiv), Et3N (1.54 mL,
11.12 mmol, 1.00 equiv), AgNO3 (2190.00 mg, 13.04 mmol, 1.17 equiv),
compound
3 (3067.9 mg, 11.12 mmol), and compound 5 (2712.0 mg,
13.29 mmol, 1.20 equiv) in acetonitrile (150 mL) was stirred under Ar at
1008C for 3.5 h. The mixture was filtered and the filtrate was poured into
water. After extraction with CHCl3, the CHCl3 layer was washed with
brine and dried over Na2SO4. Filtration and evaporation afforded a resi-
due, which was subjected to silica gel flash column chromatography (tolu-
ene/n-hexane 1:1!toluene/n-hexane/AcOEt 10/10/1) to give 6 (2.60 g,
7.37 mmol, 66.3%) as a yellow solid: 1H NMR (CDCl3, 500 MHz): d
=7.99 (d, J=8.5 Hz, 2H), 7.54 (d, J=8.2 Hz, 2H), 7.27 (s, 2H), 7.16 (d,
J=16.4 Hz, 1H), 7.10 (d, J=16.1 Hz, 1H), 2.35 (s, 6H), 1.61 ppm (s,
9H).
Detection of NO with DAF-FM DA: HCT116 human colon cancer cells
were treated in the same manner as in the confocal microscopy experi-
ments. Briefly, the cells were plated on 3 cm glass-bottomed dishes at
1.0ꢂ105 cells per dish with McCoyꢁs 5 A culture medium (2 mL) and
were then incubated at 378C in a humidified incubator (CO2, 5%, v/v)
for 2 days. The dishes were washed with D-PBS and the culture medium
was replaced with D-PBS (1 mL). The cells were treated with DAF-
FM DA (10 mm) for 15 min in the dark and were then washed twice with
D-PBS. The cells were subsequently treated with RpNO (100 mm) for
15 min in the dark, washed twice with D-PBS, and subjected to photoirra-
diation for 5 min with the light source (100 W mercury lamp) of a fluo-
rescence microscope fitted with a WU filter (330–380 nm band-pass
filter) and a 1.5% ND filter. After irradiation, the cells were subjected to
confocal fluorescence microscopy (LSM 510, Carl Zeiss Japan Co. Ltd.,
Tokyo, Japan).
Compound 7: TFA (5 mL) was added to a suspension of 6 (1355.4 mg,
3.84 mmol) in CHCl3 (5 mL). The mixture was stirred for 6.5 h at room
temperature and then concentrated in vacuo, and the residue was sus-
pended in n-hexane. Filtration gave 7 (1033.3 mg, 3.48 mmol, 90.6%) as a
yellow powder: H NMR (DMSO, 500 MHz): d=7.96 (d, J=8.2 Hz, 2H),
7.74 (d, J=8.2 Hz, 2H), 7.57 (s, 2H), 7.47 (d, J=16.4 Hz, 1H), 7.39 (d,
J=16.4 Hz, 1H), 2.30 ppm (s, 6H).
1
Compound 1 (RpNO): DMF (catalytic amount) was added to a solution
of 7 (50 mg, 0.17 mmol) and ethanedioyl dichloride (40 mL, 0.47 mmol,
2.8 equiv) in CH2Cl2 (4 mL). The mixture was stirred at room tempera-
ture for 1.5 h. Evaporation of the solvent in vacuo gave a yellow solid. A
solution of the yellow solid in CH2Cl2 (3 mL) was added at 08C to a solu-
tion of the rhodamine derivative 8[13] (137 mg, 0.25 mmol, 1.5 equiv) in
CH2Cl2 (3 mL) and Et3N (1.5 mL). The mixture was stirred at room tem-
perature for 5 h, and solvents were removed in vacuo. Purification of the
residue by silica gel flash chromatography (MeOH/CHCl3 1:15 to 1:10)
and recrystallization from MeOH/Et2O gave 1 (RpNO, 62.3 mg, 45.3%)
as a dark red solid: m.p. 185–1878C; 1H NMR (CD3OD, 500 MHz):
d=7.79–7.30 (m, 3H), 7.65 (d, J=5.0 Hz, 2H), 7.52 (m, 1H), 7.42 (s,
2H), 7.38 (d, J=8.5 Hz, 2H), 7.26 (m, 4H), 7.18 (d, J=5.0 Hz, 2H), 6.97
(s, 2H), 3.69 (q, J=7.1 Hz, 8H,), 3.44–3.07 (m, 8H), 2.30 (s, 6H),
1.31 ppm (t, J=7.5 Hz, 12H); 13C NMR (DMSO, 500 MHz): d=162.7,
160.1, 149.8, 147.7, 147.4, 130.9, 130.8, 126.9, 123.7, 122.7, 121.8, 121.7,
120.1, 119.5, 118.6, 118.5, 105.9, 105.4, 87.9, 37.4, 30.6, 16.9 ppm; ESI-MS
790 [M+HꢁCl]+; HPLC purity; 97.004%.
Cytotoxicity to HCT116 cells: HCT116 human colon cancer cells were
treated in the same manner as in the confocal microscopy experiments,
with slight modifications. In brief, the cells were plated on 6 cm culture
dishes at 2.5ꢂ105 cells per dish with McCoyꢁs 5 A culture medium (5 mL)
and incubated at 378C in a humidified incubator (CO2, 5%, v/v) for
1 day. On the day of the experiment, the culture medium was replaced
and its volume was reduced to 2 mL per 6 cm culture dish. The cells were
treated with RpNO in DMSO (20 mL) to give a final concentration of
10 mm, which resulted in a final DMSO concentration of less than 1%
AHCTUNGERTG(NNUN v/v) in the culture media; this concentration of DMSO alone had no ap-
parent effect on the appearance of the cells. The cells were also treated
with RpNO in the same manner as described above, but in the presence
of P450 nor (200 nm, Wako Pure Chemical Industry Co. Ltd, Osaka,
Japan) and b-NADH (10 mm) for scavenging. The treated cells were pho-
toirradiated for 5 min with the light source (xenon lamp) of an irradiating
apparatus (Asahi Spectra MAX-302) fitted with a UV filter (325–375 nm
band-pass filter). The irradiation was limited to a circular area of 5 mm
diameter. After 24 h incubation, the cells were observed under an invert-
Confocal microscopy: HCT116 human colon cancer cells were purchased
from American Type Culture Collection (ATCC) and cultured in
McCoyꢁs 5 A culture medium containing penicillin and streptomycin and
supplemented with fetal bovine serum according to the manufacturerꢁs
instructions. The cells were maintained at 378C in a humidified incubator
(CO2, 5%, v/v) under subconfluent conditions. For the experiments, the
cells were plated on 3 cm glass dishes at 1.0ꢂ105 cells per dish with the
culture medium (2 mL). The cells were incubated at 378C in a humidified
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Photoinduced NO Release from a Nitrobenzene Derivative in Mitochondria
FULL PAPER
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Received: July 12, 2010
Published online: March 14, 2011
Chem. Eur. J. 2011, 17, 4809 – 4813
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