Neuroprotection against superoxide anion radical by metallocorroles in cellular and murine models of optic neuropathy

Article abstract of DOI:10.1111/j.1471-4159.2010.06781.x

Corroles are tetrapyrrolic macrocycles that have come under increased attention because of their unique capabilities for oxidation catalysis, reduction catalysis, and biomedical applications. Corrole-metal complexes (metallocorroles) can decompose certain

Full text of DOI:10.1111/j.1471-4159.2010.06781.x

JOURNAL OF NEUROCHEMISTRY
|
2010
|
114
|
488–498
doi: 10.1111/j.1471-4159.2010.06781.x
,
*Maisonneuve-Rosemont Hospital Research Center and Department of Ophthalmology, University of Montreal, Montreal, Quebec,
Canada
Schulich Faculty of Chemistry, Technion – Israel Institute of Technology, Haifa, Israel
àDepartment of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Wisconsin, USA
Abstract
Serum deprivation caused increased levels of intracellular
superoxide, detected by an increase in the fluorescence
intensity of 2-hydroxyethidium, and this was blocked by Fe-
and Mn-corroles, but not Ga-corrole. In vivo real-time confocal
imaging of retinas after optic nerve transection assessed the
superoxide production within individual rat retinal ganglion
cells. Fe- and Mn-corroles, but not Ga-corrole, scavenged
neuronal superoxide in vivo. Given that the neuroprotec-
tive activity of metallocorroles correlated with superoxide
scavenging activity, Fe- and Mn-corroles could be candidate
drugs for delaying neuronal death after axonal injury in optic
neuropathies, such as glaucoma.
Corroles are tetrapyrrolic macrocycles that have come under
increased attention because of their unique capabilities for
oxidation catalysis, reduction catalysis, and biomedical appli-
cations. Corrole-metal complexes (metallocorroles) can
decompose certain reactive oxygen species (ROS), similar to
metalloporphyrins. We investigated whether Fe-, Mn-, and
Ga-corroles have neuroprotective effects on neurons and
correlated this with superoxide scavenging activity in vitro and
in vivo. Apoptosis was induced in retinal ganglion cell-5
neuronal precursor cells by serum deprivation. Cell death was
measured with sodium 3¢-[1-[(phenylamino)-carbonyl]-3,4-
tetrazolium]-bis (4-methoxy-6-nitro) benzene-sulfonic acid
hydrate and calcein-AM/propidium iodide assays. Fe- and
Mn-corroles, but not the non-redox-active Ga-corrole used as
control, reduced RGC-5 cell death after serum deprivation.
Keywords: metallocorroles, neuroprotection, neurons, super-
oxide, optic neuropathy.
J. Neurochem. (2010) 114, 488–498.
Superoxide, the result of the one electron reduction of
dioxygen, can serve as an intracellular signaling molecule for
a variety of biologically processes. It also reacts with nitric
oxide (NO) as to form peroxynitrite (HOONO), a toxin
involved in numerous diseases (Pacher et al. 2007). We
previously showed that generation of superoxide is a critical
molecular event underlying neuronal death induced by
axonal injury (Lieven et al. 2003, 2006; Nguyen et al.
2003) and in vivo imaging demonstrates that superoxide
elevation precedes apoptosis in axotomized neurons (Kana-
mori et al. in press). Intracellular delivery of pegylated
superoxide dismutase-1 (SOD), which dismutates superoxide
into H2O2, inhibits neuronal death after axotomy in vitro
(Schlieve et al. 2006) and in vivo (Kanamori et al. in press),
presumably by interfering with a superoxide signal for
apoptosis. This mechanism has therapeutic implications for
disease associated with axonal injury.
However, translation of a therapy that relies on intra-
cellular protein delivery is less practical than one that
utilizes small molecules. We therefore took advantage of
Received February 12, 2010; revised manuscript received April 15,
2010; Accepted April 19, 2010.
Address correspondence and reprint requests to Leonard A. Levin,
M.D., Ph.D., Maisonneuve-Rosemont Hospital, 5415 boul. de l’As-
somption, Montre´al, Quebec, Canada H1T 2M4 or Zeev Gross, Ph.D.,
Schulich Faculty of Chemistry, Technion City, Technion, Haifa 32,000,
Israel. E-mail: lalevin@facstaff.wisc.edu or chr10zg@tx.technion.ac.il
Abbreviations used: 2-OH-Et, 2-hydroxyethidium; BSA, bovine ser-
um albumin; CSLO, confocal scanning laser ophthalmoscopy; DiR, 1,1¢-
dioctadecyl-3,3,3¢,3¢-tetramethylindotricarbocyanine iodide; HEt, hy-
droethidine; HOONO, peroxynitrite; NO, nitric oxide; PI, propidium
iodide; RGC, retinal ganglion cell; ROS, reactive oxygen species; SOD,
superoxide dismutase; XTT, sodium 3¢-[1-[(phenylamino)-carbonyl]-3,4-
tetrazolium]-bis (4-methoxy-6-nitro) benzene-sulfonic acid hydrate.
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Table 1 Reactive oxygen species scav-
Superoxide dismutase
activity, kcat/M)1s)1
Decomposition of
peroxynitrite, kcat/M)1s)1
Catalase activity,
kcat/M)1s)1
enging by metallocorroles
Cytochrome Pulse
c assay
radiolysis
Stopped-flow
Stopped-flow
Fe(tpfc)(SO3H)2 1.7 · 106 (a) 3 · 106 (a)
3.1 · 106 (c)
4.3 · 103 (d)
ND
Mn(tpfc)(SO3H)2 4.8 · 105 (a) < 1 · 105 (a) 8.6 · 104 (c)
Mn(TMPyP)
3.8 · 106 (b) 5.6 · 106 (a) Not catalytic (data in the
presence of ascorbate
20 (f)
co-reductant: 3.3 · 104) (e)
Inactive (d)
Ga(tpfc)(SO3H)2 Inactive (d)
Inactive (d)
Inactive (d)
a, Data from Eckshtain et al. (2009); b, data from Batinic-Haberle et al. (1998); c, data from
Mahammed and Gross (2006); d, A. Mahammed and Z. Gross, unpublished data; e, data from
Crow (1999); f, data from Day et al. (1997).
recent developments in metallocorrole chemistry as a
means of using small molecules to modulate superoxide
signaling. Corroles are tetrapyrrolic macrocycles with a
porphyrin-like inner core containing four nitrogen atoms
serving as an equatorial coordination template for metal
ions. Depending on the metal, these molecules can
function as components in oxidation catalysis (Fe, Mn,
Cr), reduction catalysis (Cr, Mn, Fe), group transfer
catalysis (Rh, Fe), sensors of gaseous molecules (Co),
and medicinal research (Aviv and Gross 2007; Aviv-Harel
and Gross 2009). The latter includes research performed
on cellular and murine models of cancer (Agadjanian et al.
2006; Agadjanian et al. 2009), cardiovascular (Haber et al.
2008), diabetes (Okun et al. 2009), and neurodegenerative
diseases (Kupershmidt et al. 2010). Over the past decade
they have received increasing attention, following the
major discoveries for their facile synthesis (Gross and
Galili 1999; Gryko 2002). Porphyrins have been used in
numerous applications, including the use of metalloporphy-
rins as neuroprotective agents that work via decomposition
of ROS (Cuzzocrea et al. 2001). We predicted that
metallocorroles, some of which can be highly specific
ROS scavengers (reviewed in Table 1), might be useful
small molecule drugs for inhibiting ROS-mediated signal-
ing of cell death.
To study this, we investigated the neuroprotective
effect of iron (Fe), manganese (Mn), and gallium (Ga)
corroles (Fig. 1) in both in vitro and in vivo models
of neuronal death. For the in vitro studies, we used
serum-deprived neuronal precursor retinal ganglion cell
(RGC)-5 cells, which when differentiated with low
levels of staurosporine, have several features similar to
that of mature neurons (Frassetto et al. 2006). For in vivo
studies, we used confocal scanning laser ophthalmoscopy
(CSLO) coupled with intravitreal fluorescent dyes to
demonstrate that certain metallocorroles function as SOD
mimetics.
Fig. 1 Chemical structure of metallocorroles used in the study.
Methods
Materials
RGC-5 cells were a generous gift of Neeraj Agarwal, Ph.D.;
Dulbecco’s modified Eagle’s medium and fetal bovine serum were
from GIBCO (Grand Island, NY, USA) Staurosporine was from
Alexis Biochemicals (San Diego, CA, USA). Menadione, peni-
cillin-streptomycin and sodium 3¢-[1-[(phenylamino)-carbonyl]-
3,4-tetrazolium]-bis (4-methoxy-6- nitro) benzene-sulfonic acid
hydrate (XTT) were from Sigma-Aldrich (St. Louis, MO,
USA). Calcein-AM, propidium iodide (PI), 1,1¢-dioctadecyl-
3,3,3¢,3¢-tetramethylindotricarbocyanine iodide [DiR; DiIC18(7)],
and hydroethidine (HEt) were from Invitrogen (Eugene, OR,
USA).
Metallocorrole synthesis
The iron(III), manganese(III), and gallium(III) complexes of
2,17-bis-sulfonato-5,10,15-tris(pentafluorophenyl)corrole (Fe(tpfc)
(SO3H)2, Mn(tpfc)(SO3H)2, Ga(tpfc)(SO3H)2) were synthesized as
described previously (Saltsman et al. 2002; Mahammed and Gross
2005).
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Mn(tpfc)(SO3H)2
72 h when cells were approximately 60–75% confluent, replated at a
1 : 20 dilution in a 25 cm2 flask in 5 mL of cell culture media and
incubated at 37ꢁC in humidified 5% CO2.
A flask loaded with a 10 mL of N,N-dimethylformamide solution of
2,17-bis-sulfonato-5,10,15-tris(pentafluorophenyl)corrole (H3(tpfc)
(SO3H)2, 15 mg, 16 lmol), and Mn(OAc)2•4H2O (15 mg, 61 lmol)
was heated to reflux for 15 min, followed by evaporation of the
solvent. The inorganic salts were separated by column chromatog-
raphy on silica (eluent: ethanol), affording 15 mg (15 lmol, 94%
yield) of the manganese(III) complex of H3(tpfc)(SO3H)2. 19F NMR
(CD3OD): d = )123.0 (brs, ortho-F), )128.0 (brs, ortho-F), )131.0
(brs, ortho-F), )153.5 (s, para-F), )154.1 (s, para-F), )156.6 (s,
para-F), )159.5 (s, meta-F), )162.9 (s, meta-F). UV/vis (buffer
solution, pH 7.30) kmax [e(M)1 cm)1)] = 392 (19 000), 422
(21 000), 480 (17 000), 644 (11 500), 610 (9500), 576 (9000). MS
(matrix-assisted laser desorption/ionization-time of flight mass
spectrometry): m/z 1007.9 [M+, 100%], 1008.9 [MH+, 85%].
Treatments
RGC-5 cells were seeded onto 6-well or 96-well microplates and
pre-incubated for 24 h. Differentiation was induced with stauro-
sporine (316 nM) for 4 h. RGC-5 cells were then serum deprived for
48 h in the presence or absence of metallocorroles.
Assessment of cell viability
XTT assay
XTT assays were used to determine the effects of treatment on cell
viability. XTT, a tetrazolium salt, is cleaved by dehydrogenation in
metabolically active cells, yielding a highly colored, water-soluble
formazan product. In non-dividing cells, where there is no
proliferation, the XTT signal is essentially proportional to the
number of viable cells. Unlike other tetrazolium salts, such as (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, XTT
does not require solubilization of formazan crystal prior to
absorbance measurements. The XTT labeling and electron coupling
reagents were added to treated serum-deprived or non-deprived cells
4 h before spectrophotometric analysis. All XTT assays were
performed in 96-well microplates. Optical density was measured
on a microplate reader (BioTek ELx808, Winooski, VT, USA) with
a 490-nm test wavelength and a 630-nm reference wavelength.
Negative controls (no cells) and positive controls (cells without
serum deprivation) were assayed in parallel. N = 8 per condition.
Experiments were performed in triplicate.
Ga(tpfc)(SO3H)2
A solution of H3(tpfc)(SO3H)2 (20 mg, 21 lmol) in pyridine
(10 mL) was added to a flask that contains a large excess (about
0.2 g) of flame-dried GaCl3, and the reaction mixture was heated to
reflux for 30 min under argon, followed by evaporation of the
solvent. The product was dissolved in 75 mL basic water (0.1 M
Na2CO3) and washed with three portions of dichloromethane. The
water was evaporated and the inorganic salts were separated
by column chromatography on silica (eluent, ethanol), affording
19 mg (19 lmol, 90% yield) of the gallium(III) complex of
H3(tpfc)(SO3H)2. 1H NMR (CD3OD): d = 9.89 (s, 1H), 8.84 (s,
1H), 8.78 [d, 3J(H,H)] 4.8 Hz, 1H), 8.65 (d, 3J(H,H) = 4.8 Hz, 1H),
8.55 (t, 3J(H,H) = 4.3 Hz, 2H). 19F NMR (CD3OD): d = )139.0 (d,
3J(F,F) = 23.0 Hz, 2F, ortho-F), )140.4 (d, 3J(F,F) = 23.5 Hz, 4F,
ortho-F), )157.6 (t, 3J(F,F) = 20.1 Hz, 1F, para-F), )158.2 (t,
3J(F,F) = 20.5 Hz, 1F, para-F), )160.2 (t, 3J(F,F) = 20.3 Hz, 1F,
para-F), )166.3 (m, 4F, meta-F), )169.1 (m, 2F, meta-F). UV/vis
(buffer solution, pH 7.30) kmax [e(M)1 cm)1)] = 424 (75 000), 588
(13 600), 610 (17 300). MS (matrix-assisted laser desorption/
ionization-time of flight mass spectrometry): m/z 1022.2 [M+,
100%] and a characteristic isotopic pattern of 1023.2 (55%), 1024.2
(92%), 1025.2 (58%). MS (electrospray): m/z 509.9 [(M ) 2H)/2]).
Fluorescent live-dead assays
RGC-5 cells were seeded onto coverslips in six-well microplates.
After treatment with metallocorroles or vehicle for 48 h in serum
deprivation, media was aspirated from wells and cells stained with
1 lM calcein-AM and 4 lM PI in phosphate-buffered saline (PBS)
for 30 min. The staining solution was aspirated and replaced with
PBS. Cells were fixed with 4% paraformaldehyde and coverslips
mounted on slide glasses using PermaFluor (Thermo Scientific,
Walthan, MA, USa). Slides were photographed under epifluores-
cence and digitized. Three visual fields were randomly sampled
from each well, at least 50 cells were counted from each field, and
three wells were analyzed for each condition. Live (calcein-positive)
and dead (PI-positive) cells were manually counted by an observer
masked to the treatment group.
Fe(tpfc)(SO3H)2
One portion of FeCl2•4H2O (55 mg, 277 mmol) was added at once
to a pyridine solution (20 mL) of H3(tpfc)(SO3H)2 (240 mg,
251 lmol), and the mixture was heated immediately to reflux for
5 min under argon, followed by evaporation of the solvent. The
inorganic salts were separated by column chromatography on silica
(eluent: ethanol), affording 240 mg (238 lmol, 95% yield) of the
iron(III) complex of H3(tpfc)(SO3H)2. 19F NMR (CD3OD):
d = )106.2 (brs, ortho-F), )115.4 (brs, ortho-F), )116.5 (brs,
ortho-F), )149.8 (s, para-F), )150.5 (s, para-F), )154.5 (s, para-
F), )156.2 (s, meta-F), )157.2 (s, meta-F), )160.2 (s, meta-F).
UV-vis (buffer solution, pH 7.00) kmax [e(M)1 cm)1)] = 404
(34 000), 552 (12 000), 738 (2300). MS (electrospray): m/z 503.5
[(M ) 2H)/2]).
Measurement of intracellular superoxide
Cells were assessed for the production of superoxide by visualizing
the reaction of HEt with superoxide, which forms 2-hydroxyethi-
dium (2-OH-Et). HEt is a non-fluorescent, reduced form of
ethidium that can passively cross plasma membranes of live cells.
When HEt is oxidized to 2-OH-Et by superoxide, it can bind to
DNA and yield fluorescence in the red spectrum (excitation
480 nm/emission 567 nm) (Zhao et al. 2003). Assays were
performed in six-well microplates in triplicate. After 48 h of
serum deprivation in the presence or absence of metallocorrole,
HEt (50 lM) was added to the medium of cells 30 min before the
end of the serum deprivation period to visualize superoxide.
Cell culture
RGC-5 cells were cultured in Dulbecco’s modified Eagle’s medium
containing 1 g/L glucose, 10% fetal bovine serum, 100 U/mL
penicillin, and 100 lg/mL streptomycin. Cells were split every 48–
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Neuroprotective metallocorroles
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Incubation of cells with menadione (1 mM) for 30 min was used as
a positive control for production of superoxide. After incubation of
HEt, cells were washed with PBS and digitally captured by
quantitative epifluorescence microscopy. Three wells were tested
for each group and three microscope fields were randomly sampled
from each well at the same exposure setting. The excitation spectra
of 2-OH-Et and other HEt oxidation products are similar at
medium wavelengths (540–580 nm), but differ at short wave-
lengths (370–400 nm), allowing the use of different excitation
filters in ex vivo fluorescence microscopy to distinguish the two
species (Robinson et al. 2006). Cells that fluoresce when excited at
395 nm are 2-OH-Et-positive, while cells fluorescing when excited
at 560 nm are positive for all HEt oxidation products. We therefore
used two filter sets for HEt fluorescent microscopy. A 395
5.5 nm excitation, 500 nm dichroic, and 562 20 nm emission
filter was used to selectively image 2-OH-Et, and a 560 20 nm
excitation, 595 nm dichroic, and 630 30 nm emission was used
to non-selectively image all HEt oxidation products. ImageJ
(National Institutes of Health, Bethesda, MD, USA) was used to
quantify the HEt signals. Briefly, cells in captured fields were
segmented by Set Threshold From Background. Mean fluorescence
intensity and granularity (standard deviation of all pixel intensities)
of single cells were then evaluated by Analyze Particle.
(10 nM–100 lM) of metallocorroles in cultures of the RGC-
5 neuronal precursor cell line in the presence of serum for
48 h. Fe-, Mn-, and Ga-corroles had no effect on viability of
undifferentiated and differentiated RGC-5 cells at concen-
trations of £ 1 lM (Fig. 2). Cell death ranged from 13% to
40% with 10 lM concentrations of all metallocorroles in the
presence of serum. The toxicity of the metallocorroles at
10 lM was significantly greater in the absence of serum,
which induced 37–80% cell death (Fig. 2b).
The greater toxicity of metallocorroles when serum was
absent made it difficult to determine their capacity for
preventing or delaying neuronal death in a serum deprivation
model. We therefore tested several serum and serum-free
conditions to find a functional paradigm for serum depriva-
tion without significant metallocorrole toxicity. Bovine
serum albumin (BSA) at a concentration of 10 mg/mL did
not affect RGC-5 survival rates in the presence of 1% serum,
while it dramatically decreased the toxicity of metallocor-
roles in the absence of serum. For example, the survival rate
of undifferentiated cells with 10 lM Fe-corrole was
increased from 29 1.7% to 63 4.4% (p < 0.001) in the
presence of 1% BSA (Fig. 2b).
Measurement of superoxide in vivo
To test the effect of metallocorroles on superoxide in vivo, the
optic nerve was intraorbitally transected in Long-Evans rats as
described (Kanamori et al. 2010) and levels of superoxide studied
by intravitreal injection of HEt followed by in vivo CSLO. To
confirm the localization of the product of superoxide with HEt,
RGCs were retrogradely labeled by stereotactically injecting
2.1 lL of DiR (20 mg/mL) into each superior colliculus of rats
5 days before optic nerve transection (Kanamori et al. in press).
Axotomy by optic nerve transection causes apoptosis of RGCs.
HEt or corrole + HEt was injected into the vitreous 3 days after
transection. The vitreous concentration of HEt and corrole were
100 lM and 100 nM, respectively, based on a vitreous volume in
the rat of 56 lM (Berkowitz et al. 1998). The next day, retinal
imaging by CSLO was performed under ketamine/xylazine
anesthesia. Pupils were dilated with phenylephrine/atropine. Ret-
inal images were obtained using a 30ꢁ field of view and real-time
averaging of at least 50 images on a Heidelberg Retina Angiograph
II (Heidelberg Engineering, Germany) CSLO at 93% sensitivity.
The plane of focus was adjusted to the inner retina by imaging the
nerve fiber layer at 488 nm.
Metallocorroles prevent neuronal cell death from serum
deprivation
Two different approaches were taken to evaluate the
neuroprotective effects of metallocorroles. First, to compare
the protective efficacy of different metallocorroles, undiffer-
entiated or differentiated RGC-5 cells were exposed to serum
deprivation with and without 1% BSA for 48 h in the
presence of varying concentrations (1 nM–10 lM) of each
metallocorrole. Cell viability was determined by XTT assay.
Serum deprivation significantly reduced the viability of
RGC-5 cells to approximately 60% of control. Addition of
Fe- and Mn-corroles rescued these cells in a dose-dependent
fashion with an optimal concentration range of 10–100 nM
in the absence of serum (Fig. 3). Ga-corrole was not
neuroprotective at any concentration in RGC-5 cells. The
neuroprotective effects of Fe- and Mn-corroles were greater
in the presence of 1% BSA (Fig. 3), presumably by
decreasing metallocorrole toxicity that was exacerbated by
the absence of protein.
To demonstrate that these results were not confounded by
the method used to determine viability, i.e. the chemical
reduction of XTT, a redox-independent viability assay was
also used, i.e. calcein-AM/PI (live/dead) double staining.
In undifferentiated RGC-5 cells, there were very few PI-
positive cells in control cultures (Fig. 4). Serum deprivation
significantly increased the proportion of PI-positive cells from
1.0 0.3% to 5.2 0.1% (p < 0.005). Addition of Fe-corrole
at concentrations of 10 nM or 100 nM to serum-deprived cells
significantly decreased the proportion of PI-positive cells
(10 nM: 2.3 1.1%, p = 0.03 vs. control; 100 nM: 1.7
Statistics
Viability was assessed as percentage of control, and compared with
untreated controls within each experiment. Means were compared
using Student’s unpaired t-test. All data are shown as mean SEM.
Significant difference required p < 0.05.
Results
Metallocorrole toxicity
Given that the toxicity of metallocorroles on RGC-5 cells
was unknown, we tested a wide range of concentrations
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492
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(a)
(b)
Fig. 2 Metallocorroles are toxic only at high concentrations. (a)
Undifferentiated and differentiated retinal ganglion cell (RGC)-5 cells
in complete media (10% serum) were treated with increasing con-
centrations of Fe, Mn, and Ga-corroles. Cell survival was measured by
sodium 3¢-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis (4-meth-
oxy-6- nitro) benzene-sulfonic acid hydrate assay. *p < 0.05 between
the samples treated with and without metallocorroles. (b) RGC-5 cells
were treated with or without a high (10 lM) concentration of Fe, Mn,
and Ga-corroles in complete (10% serum) media (stippled bars),
serum-deprived media (closed bars) and serum-deprived media con-
taining 1% bovine serum albumin (BSA) (open bars). BSA decreased
the toxicity of metallocorroles in the absence of serum. SD, serum
deprivation, *p < 0.05.
0.2%, p = 0.002 vs. control). Similarly, Mn-corrole at
concentrations of 10 nM or 100 nM decreased PI-positivity
(10 nM: 1.3 0.1%, p < 0.001 vs. control; 100 nM: 1.5
0.5%, p = 0.002 vs. control) (Fig. 4b). On the other hand,
there was no significant difference in PI-positivity in serum-
deprived cells treated with and without Ga-corrole, indicating
an absence of neuroprotection. All metallocorroles at 10 lM
increased the number of PI-positive cells, indicating toxicity at
that concentration.
In RGC-5 cells differentiated with staurosporine, the
proportion of PI-positive cells in normal conditions was
6.6 0.8%, suggesting a baseline level of apoptosis from
the treatment. This increased with serum deprivation to
35 2.1%. Fe and Mn-corroles but not Ga-corrole decreased
the proportion of PI-positive cells at concentrations of 10 and
100 nM [Fe (10 nM): 16 5.6%, p = 0.03; Fe (100 nM):
18 1.2%, p = 0.002; Mn (10 nM): 32 1.5%, p = 0.031;
Mn (100 nM): 31 2.9%, p = 0.041).
Serum deprivation induces superoxide production
Intracellular superoxide was visualized by imaging fluores-
cence of 2-OH-Et, the product of HEt and superoxide
(Fig. 5). Excitation filters that were selective (395 nm) or
non-selective (560 nm) for 2-OH-Et were used to generate
epifluorescent images of serum-deprived cells. The fluores-
cence intensity seen with 2-OH-Et-selective excitation was
less than with the non-selective excitation because the
bandwidth of the former filter was much narrower than the
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Neuroprotective metallocorroles
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(a)
(b)
Fig. 3 Undifferentiated (left) or differenti-
ated (right) retinal ganglion cell (RGC)-5
cells serum-deprived in 1% bovine serum
albumin (BSA) were treated with increasing
concentrations of Fe-, Mn-, or Ga-corroles.
The rescue from serum deprivation was
measured by sodium 3¢-[1-[(phenylamino)-
carbonyl]-3,4-tetrazolium]-bis (4-methoxy-
6-nitro) benzene-sulfonic acid hydrate
assay, normalized to 100% rescue with
serum. Closed and open bars show serum
deprivation (SD) and SD with 1% BSA,
respectively. *p < 0.05 between the sam-
ples treated with and without metallocor-
roles in SD. These data are summarized
from three independent experiments.
latter (11 vs. 40 nm). The mean intensity per cell was used to
measure total amount of superoxide, and granularity (stan-
dard deviation of all pixels within a cell) of intensity used
to indicate dispersion of superoxide from subcellular com-
partments. Dying cells with shrinkage of cell body had high
granularity. In both undifferentiated and differentiated
RGC-5 cells, serum deprivation increased the fluorescence
intensity and granularity, compared with serum-containing
controls. Fe- and Mn-corroles (100 nM) significantly sup-
pressed the increase of superoxide in undifferentiated and
differentiated RGC-5 cells. Ga-corrole (100 nM) did not
have a significant effect on superoxide levels. Menadione
(1 mM), used as a positive control for superoxide induction,
resulted in a burst of 2-OH-Et in both undifferentiated and
differentiated RGC-5 cells. To prove that superoxide and not
another ROS was being imaged, cells were incubated with
polyethylene glycol-conjugated superoxide dismutase, which
crosses cell membranes and dismutates superoxide into
hydrogen peroxide. Polyethylene glycol-conjugated super-
oxide dismutase (30 U/mL) reduced 2-OH-Et levels in
serum-deprived differentiated cells from 133.2 5.7 to
75.1 4.4 arbitrary light units (p < 0.001).
Metallocorroles scavenge axotomy-induced superoxide
in vivo
In vivo detection of superoxide in RGCs after axotomy was
carried out by CSLO after intravitreal injection of HEt. Two
built-in CSLO lasers were utilized. The 488 nm laser was
used for excitation of 2-OH-Et and the 788 nm laser was
used for excitation of DiR. Cells positive for 2-OH-Et after
optic nerve transection could be localized to the ganglion cell
layer by confocal focusing on the innermost retina, and
identified as being in RGCs by colocalization with the
retrogradely transported fluorescent dye DiR (Fig. 6a–c).
CSLO images of eight retinal quadrants immediately adja-
cent to the optic nerve head were obtained at each session,
and positive cells counted (Fig. 6e). Optic nerve transection
increased the number of 2-OH-Et-positive cells (Fig. 6f).
Fluorescence was absent in the outer retina and retinas with
untransected optic nerves. The peak of 2-OH-Et positivity
was seen at 4 days after axotomy (66.2 11.6 cells/retina).
Therefore, we counted the number of 2-OH-Et-positive cells
at 4 days after transection in retinas treated with (n = 4) and
without metallocorrroles (n = 8). Fe- and Mn-corroles
(100 nM), but not Ga-corrole, significantly decreased the
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494
|
A. Kanamori et al.
(a)
(b)
Undifferentiated RGC-5
*
Differentiated RGC-5
100
100
80
60
40
20
0
*
PI
Calcein + PI
Magnified insets
95
90
5
*
*
*
*
*
*
Serum
Fe-corrole
+
–
–
–
–
–
–
–
Serum
+
–
–
–
–
–
–
–
10 nM 100 nM1 µM 10 µM
Fe-corrole
10 nM 100 nM1 µM 10 µM
*
100
55
*
50
*
80
60
40
20
45
40
*
*
5
*
*
*
*
Serum0
Fe-corrole
Serum
Mn-corrole
+
–
–
–
–
–
–
–
+
–
–
–
–
–
–
–
10 nM 100 nM1 µM 10 µM
10 nM 100 nM1 µM 10 µM
*
100
90
5
100
80
60
40
20
*
*
Serum0
Fe-corrole
+
–
–
–
–
–
–
–
Serum
Ga-corrole
+
–
–
–
–
–
–
–
10 nM 100 nM1 µM 10 µM
10 nM 100 nM1 µM 10 µM
Fig. 4 (a) Representative photomicrographs of retinal ganglion cell
(RGC)-5 cells stained with calcein-AM/propidium iodide (PI). Undif-
ferentiated or differentiated RGC-5 cells were treated with either ser-
um deprivation without metallocorroles or serum deprivation with
100 nM Fe-corrole. (Left) Propidium iodide (red) staining of dead cells.
(Center) Merge of propidium iodide and calcein (green). Green stain-
ing indicates live cells and yellow indicates late apoptotic/necrotic
cells. (Right) Magnified insets from outlined fields in center column.
Scale bar, 20 lm. SD, serum deprivation. (b) Summary of calcein-AM/
PI cell-survival assay data. Quantitative data from fluorescence ima-
ges derived from the calcein/PI cell-survival assay are expressed as
the mean SEM of results from three wells in each condition. Three
microscope fields containing more than 50 cells were randomly sam-
pled from each well. *p < 0.05 compared with serum-deprived RGC-5
without metallocorroles.
number of superoxide-positive cells/retina (Fe: 20 11.0,
p = 0.028; Mn: 22.5 6.8, p = 0.013; Ga: 49.5 5.9,
p = 0.26), as shown in Fig. 6f.
idyl)porphyrin pentachloride, Mn-corrole has slightly lower
levels of SOD activity, and Ga-corrole is inactive (Eckshtain
et al. 2009). Fe- and Mn-corrole shorten the half-life of
HOONO, the product of superoxide and NO, very signifi-
cantly (Fe > Mn), and Ga-corrole is inactive (Mahammed
and Gross 2006). Fe-corrole but not Ga-corrole catalyzes the
decomposition of H2O2. These and other ROS are critical for
specific types of neuronal death. Superoxide is necessary for
the signaling of apoptosis after axonal injury (Lieven et al.
2003; Kanamori et al. in press), while HOONO is a potent
initiator of neuronal cell death and is linked to several
neurodegenerative disorders (Torreilles et al. 1999).
Some metalloporphyrins, particularly Fe- and Mn-porphy-
rins, mimic SOD and decompose superoxide and HOONO
quite efficiently. Neuroprotective effects of Mn-porphyrins as
SOD mimetics have been demonstrated in animal models of
ischemia (Salvemini et al. 1999; Mackensen et al. 2001;
Sheng et al. 2002). Fe-porphyrins acting as HOONO
decomposition catalysts protect cytokine-induced cyto-
toxicity in hippocampal cultures (Misko et al. 1998) and
Discussion
These results demonstrate that (i) Fe- and Mn-corroles but
not Ga-corrole significantly reduce cell death induced by
serum deprivation in neuronal precursor cells; (ii) Fe- and
Mn-corroles but not Ga-corrole significantly decrease super-
oxide levels in tissue culture; (iii) Fe- and Mn-corroles
significantly decrease superoxide levels in RGCs after
axotomy in vivo. Together, these findings suggest that not
only are metallocorroles a novel class of neuroprotectants,
but that the mechanism of action covaries with a reduction in
intracellular superoxide levels in vitro and in vivo.
Studies in purely chemical systems demonstrate that
metallocorroles are powerful ROS scavengers (Table 1).
Fe-corrole is a potent SOD mimetic, with similar activity to
the extensively studied (Mn(III)tetrakis(1-methyl-4-pyr-
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Neuroprotective metallocorroles
| 495
(a)
(b)
(c)
Fig. 5 (a) Undifferentiated and differentiated retinal ganglion cell
(RGC)-5 cells were incubated with hydroethidine (HEt) to visualize
superoxide. Serum-deprived RGC-5 cells were treated with metal-
locorroles at 100 nM or media control. Images were taken with
either a non-selective filter set that detected oxidative products of
HEt (pseudocolored red) or a filter set selective for 2-hydroxyethi-
dium (2-OH-Et) (pseudocolored yellow). The inset shows magnified
cells with high granularity (left) and low granularity (right). (b and c)
Fluorescence intensity and granularity in undifferentiated (b) and
differentiated (c) RGC-5 cells were calculated with ImageJ.
Polyethylene glycol-conjugated superoxide dismutase (30 U/mL)
was added in the absence of serum. Menadione (1 mM) was used
in the presence of serum to induce mitochondrial superoxide
production by redox cycling. Bars indicate results with filters non-
selective (closed bars) and selective (open bars) for 2-OH-Et.
*p < 0.001 compared with serum control condition. **p < 0.001
compared with serum-deprived condition. ***p < 0.05 compared with
serum-deprived condition.
methamphetamine-induced neurotoxicity in rats (Imam et al.
2001). One potentially problematic chemical feature com-
mon to both metallocorroles and metalloporphyrins is that
they may use ROS (e.g., hydrogen peroxide) for either
catalyzing oxidation of other substrates (i.e., serving as pro-
oxidants) or decomposing them (i.e., serve as anti-oxidants).
One advantage of metallocorroles relative to metalloporphy-
rins is that their antioxidant property is more pronounced
than their pro-oxidant capability.
Okun et al. 2009). Some reported metallocorroles have high
water-solubility and amphiphilicity, which distinguishes
them from most metalloporphyrins. In addition, Fe-, Mn-,
and Ga-corroles form tightly bound non-covalent conju-
gates with variety of proteins, including serum albumins
(Mahammed et al. 2004; Haber et al. 2004). In this study,
coincubation with 1% BSA significantly decreased the
toxicity of high concentrations of these metallocorroles on
serum-deprived RGC-5 cells.
Metallocorroles have also other properties that make them
more useful for certain purposes than metalloporphyrins,
such as the intense fluorescence of gallium corroles (Liu
et al. 2008; Kowalska et al. 2009) that has been used for
cellular and whole animal imaging (Agadjanian et al. 2009;
We studied both undifferentiated RGC-5 cells and RGC-5
cells differentiated with staurosporine. Undifferentiated
RGC-5 cells are similar to neuronal precursor cells, dividing
in culture and expressing an immature neuroglial morphol-
ogy. Differentiated RGC-5 cells are post-mitotic, extend
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496
| A. Kanamori et al.
(a)
(b)
(c)
Fig. 6 (a–c) In vivo imaging confirmation
that retinal ganglion cells express 2-hy-
droxyethidium (2-OH-Et) after optic nerve
transection (ONT). (a) RGCs positive for
1,1¢-dioctadecyl-3,3,3¢,3¢-tetramethylindotri-
carbocyanine iodide retrogradely trans-
ported from the superior colliculi were
excited by the 788 nm laser. These cells
are pseudocolored green. (b) 2-OH-Et-po-
sitive cells excited by the 488 nm laser after
intravitreal administration of hydroethidine
(HEt). These cells are pseudocolored red.
(c) Merged image of (a) and (b). (d) Flow-
chart of experiments. (e) Schematic of
topography of retinal imaging by confocal
scanning laser ophthalmoscopy. To quan-
tify the number of cells positive for 2-OH-Et,
eight images were taken at each retinal
octant adjacent to the optic nerve head. (f)
The number of HEt-positive ganglion cell
layer cells at 4 days after optic nerve tran-
section in the presence or absence of Fe-,
Mn-, and Ga-corroles. *p < 0.05.
(d)
(e)
(f)
neurites with characteristics of dendrites and axons, and
therefore have the advantage of being similar to mature
neurons. On the other hand, the drug used to differentiate
RGC-5 cells, staurosporine, can also induce cytochrome c
release (Lieven C. J. and Levin L. A., unpublished data) and
low levels of baseline PI staining (Fig. 4), suggesting that
some differentiated cells are undergoing apoptosis in our
culture conditions. Because there are advantages and disad-
vantages for using both undifferentiated and differentiated
cells, we studied the effects of metallocorroles in both
models. Despite the differences in differentiation status, the
superoxide scavenging and neuroprotective effects of Fe- and
Mn-corroles were the same.
Our results indicate that the Fe- and Mn-corroles used in
this study are able to decrease intracellular superoxide levels
in neuronal precursor cells in vitro and retinal neurons
in vivo. Serum deprivation was used in this study to induce
neuronal cell death, via deprivation of molecules necessary
for survival, including neurotrophic factors. One of the
mechanisms by which neurotrophin deprivation causes
apoptosis of neuronal cells is initiating production of
intracellular ROS (Greenlund et al. 1995; Kirkland et al.
2007). We previously demonstrated that a critical molecular
event underlying neuronal death after in vitro and ex vivo
models of axonal injury is the generation of superoxide anion
(Lieven et al. 2003, 2006; Nguyen et al. 2003). Real-time
in vivo confocal imaging demonstrates that superoxide
elevation precedes apoptosis in RGCs after optic nerve
transection (Kanamori et al. in press) Using a similar in vivo
imaging system in this study allowed us to measure
intracellular superoxide within retinal neurons, based on
the fluorescent product of superoxide with HEt. When HEt
oxidation products are excited at 560 nm, both the superox-
ide-HEt product 2-OH-Et and the peroxide-HEt oxidation
product Et can be visualized (Robinson et al. 2006). In our in
vivo experiments, the 488 nm CSLO laser was used for
excitation. We previously showed (Kanamori et al. in press)
that the fluorescence detected with the CSLO with 488 nm
excitation is mainly generated by the 2-OH-Et product
(which is specific to superoxide), but there can also be
fluorescence from Et, which is partially excited at that
wavelength. To address this possibility, we previously
correlated the in vivo imaging findings with histological
examination of the same retinas using 395 nm excitation by
epifluorescence microscopy (Kanamori et al. in press). These
studies confirmed that the HEt-positivity detected in vivo
arose from the superoxide product 2-OH-Et.
Using the CSLO for imaging of RGCs in living animals,
we were able to show that intravitreal administration of Fe
and Mn-corroles decreased retinal superoxide in vivo. This
was supported by our finding that Fe- and Mn-corroles
decrease superoxide levels in RGC-5 cells induced by serum
deprivation. Together, these results suggest that Fe- and Mn-
corroles may be useful as SOD mimetics for neuroprotective
diseases associated with axonal injury.
Consistent with expectations, we showed that only Fe- and
Mn-corroles, but not the redox-inactive Ga-corrole, were
both neuroprotective and decomposed superoxide. We can-
not exclude the possibility that catalytic decomposition of
another ROS besides superoxide was the critical factor for
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Journal Compilation ꢀ 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 488–498

Neuroprotective metallocorroles
| 497
metallocorrole-mediated neuroprotection. Although we used
a well-characterized model in which superoxide is known to
both be generated and is necessary for RGC death, it could
be that scavenging of HOONO (the product of superoxide
and NO) contributed to increased neuronal survival in vitro at
the same time that superoxide was being scavenged. Our
previous work failed to find evidence for NO elevation in
axotomized RGCs (Kanamori et al. in press), but perhaps a
basal level of NO could combine with elevated superoxide to
induce cell death. Measurements of ROS scavenging
(Table 1) reveals that Mn-corrole is a significantly poorer
HOONO scavenger than Fe-corrole, yet our assays demon-
strated that these metallocorroles were almost equally
neuroprotective of serum-deprived neuronal cells. Nonethe-
less, without more specific scavengers or a method for
imaging HOONO with high specificity, the possibility that
decomposition of HOONO is responsible for neuroprotection
in this paradigm cannot be completely excluded.
References
Agadjanian H., Ma J., Rentsendorj A., Valluripalli V., Youn Hwang
J., Mahammed A., Farkas D. L., Gray H. B., Gross Z. and
Medina-Kauwe L. K. (2009) Tumor detection and elimination
by a targeted gallium corrole. Proc. Nat. Acad. Sci. 106, 6100–
6105.
Agadjanian H., Weaver J. J., Mahammed A. et al. (2006) Specific
delivery of corroles to cells via noncovalent conjugates with viral
proteins. Pharm. Res. 23, 367–377.
Aviv I. and Gross Z. (2007) Corrole-based applications. Chem. Commun.
(Camb.) 1987–1999.
Aviv-Harel I. and Gross Z. (2009) Aura of corroles. Chemistry 15, 8382–
8394.
Batinic-Haberle I., Benov L., Spasojevic I. and Fridovich I. (1998) The
ortho effect makes manganese(III) meso-tetrakis(N-methylpyridi-
nium-2-yl)porphyrin a powerful and potentially useful superoxide
dismutase mimic. J. Biol. Chem. 273, 24521–24528.
Berkowitz B. A., Lukaszew R. A., Mullins C. M. and Penn J. S. (1998)
Impaired hyaloidal circulation function and uncoordinated ocular
growth patterns in experimental retinopathy of prematurity. Invest.
Ophthalmol. Vis. Sci. 39, 391–396.
There are few studies of metallocorroles as cytoprotec-
tants in vitro or in vivo. Haber et al. (2008) compared oral
Fe-, Mn-, and Ga-corroles in a mouse model of athero-
sclerosis. They found that Fe-corrole dramatically pre-
vented the development of atherosclerotic lesions and
that Mn-corrole moderately reduced the size of lesion.
Ga-corrole had no effect on the lesions despite reducing
blood cholesterol levels (Fe: 40%, Mn: 26%, Ga: 20%).
Kupershmidt et al. (2010) showed that Fe- and Mn-corroles
increased survival of human neuroblastoma and mouse
motor neuron-neuroblastoma fusion cells when treated with
3-morpholinosydnonimine, which generates HOONO, or
with 6-hydroxydopamine, which generates a variety of
ROS. Our findings indicate that Fe- and Mn-corroles could
be candidate therapeutic agents for diseases associated with
axonal injury where superoxide may be a critical factor.
Such diseases include optic neuropathies such as glaucoma,
where treatment does not always prevent progression, and
non-arteritic anterior ischemic optic neuropathy, where
there is no effective treatment. The use of specific
metallocorroles may make possible the treatment of such
diseases and potentially other superoxide-associated condi-
tions, such as physiological aging.
Crow J. P. (1999) Manganese and iron porphyrins catalyze peroxynitrite
decomposition and simultaneously increase nitration and oxidant
yield: implications for their use as peroxynitrite scavengers in vivo.
Arch. Biochem. Biophys. 371, 41–52.
Cuzzocrea S., Riley D. P., Caputi A. P. and Salvemini D. (2001) Anti-
oxidant therapy: a new pharmacological approach in shock,
inflammation, and ischemia/reperfusion injury. Pharmacol. Rev.
53, 135–159.
Day B. J., Fridovich I. and Crapo J. D. (1997) Manganic porphyrins
possess catalase activity and protect endothelial cells against
hydrogen peroxide-mediated injury. Arch. Biochem. Biophys. 347,
256–262.
Eckshtain M., Zilbermann I., Mahammed A., Saltsman I., Okun Z.,
Maimon E., Cohen H., Meyerstein D. and Gross Z. (2009)
Superoxide dismutase activity of corrole metal complexes. Dalton
Trans. 7879–7882.
Frassetto L. J., Schlieve C. R., Lieven C. J., Utter A. A., Jones M. V.,
Agarwal N. and Levin L. A. (2006) Kinase-dependent differenti-
ation of a retinal ganglion cell precursor. Invest. Ophthalmol. Vis.
Sci. 47, 427–438.
Greenlund L. J., Deckwerth T. L. and Johnson E. M. (1995) Super-
oxide dismutase delays neuronal apoptosis: a role for reactive
oxygen species in programmed neuronal death. Neuron 14, 303–
315.
Gross Z. and Galili N. (1999) N-Substituted Corroles: A Novel Class of
Chiral Ligands. Angew Chem. Int. Ed. Engl. 38, 2366–2369.
Gryko D. T. (2002) Recent advances in the synthesis of corroles and
core-modified corroles. Eur. J. Org. Chem. 2002, 1735–1743.
Haber A., Agadjanian H., Medina-Kauwe L. K. and Gross Z. (2008)
Corroles that bind with high affinity to both apo and holo trans-
ferrin. J. Inorg. Biochem. 102, 446–457.
Acknowledgements
Canadian Institutes for Health Research, Canadian Foundation for
Innovation, Canadian Research Chairs program, Fonds de recherche
en ophtalmologie de l’Universite´ de Montre´al, National Institutes of
Health. Work at the Technion was supported by the Herbert Irving
Cancer and Atherosclerosis Research Fund and the Israel Science
Foundation.
Haber A., Mahammed A., Fuhrman B., Volkova N., Coleman R., Hayek
T., Aviram M. and Gross Z. (2008) Amphiphilic/Bipolar metallo-
corroles that catalyze the decomposition of reactive oxygen and
nitrogen species, rescue lipoproteins from oxidative damage, and
attenuate atherosclerosis in mice. Angew Chem. Int. Ed. Engl. 47,
7896–7900.
Imam S. Z., el-Yazal J., Newport G. D., Itzhak Y., Cadet J. L., Slikker
W. Jr and Ali S. F. (2001) Methamphetamine-induced dopami-
nergic neurotoxicity: role of peroxynitrite and neuroprotective role
of antioxidants and peroxynitrite decomposition catalysts. Ann. NY
Acad. Sci. 939, 366–380.
Author disclosure statement
No competing financial interests exist.
ꢀ 2010 The Authors
Journal Compilation ꢀ 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 488–498

498
| A. Kanamori et al.
Kanamori A., Catrinescu M. M., Traistaru M., Beaubien R. and Levin
L. A. (2010) In vivo imaging of retinal ganglion cell axons
within the nerve fiber layer. Invest. Ophthalmol. Vis. Sci. 51,
2011–2018.
Kanamori A., Catrinescu M., Kanamori N., Mears K. A., Beaubien R.
and Levin L. A. (in press) Superoxide is an associated signal for
apoptosis in axonal injury. Brain, in press.
Mahammed A., Gray H. B., Weaver J. J., Sorasaenee K. and Gross Z.
(2004) Amphiphilic corroles bind tightly to human serum albumin.
Bioconjug. Chem. 15, 738–746.
Misko T. P., Highkin M. K., Veenhuizen A. W., Manning P. T., Stern
M. K., Currie M. G. and Salvemini D. (1998) Characterization of
the cytoprotective action of peroxynitrite decomposition catalysts.
J. Biol. Chem. 273, 15646–15653.
Kirkland R. A., Saavedra G. M. and Franklin J. L. (2007) Rapid
activation of antioxidant defenses by nerve growth factor
suppresses reactive oxygen species during neuronal apoptosis:
evidence for a role in cytochrome c redistribution. J. Neurosci.
27, 11315–11326.
Kowalska D., Liu X., Tripathy U., Mahammed A., Gross Z., Hirayama
S. and Steer R. P. (2009) Ground- and excited-state dynamics
of aluminum and gallium corroles. Inorg. Chem. 48, 2670–
2676.
Kupershmidt L., Okun Z., Amit T., Mandel S., Saltsman I., Mahammed
A., Bar-Am O., Gross Z. and Youdim M. B. (2010) Metallocor-
roles as cytoprotective agents against oxidative and nitrative stress
in cellular models of neurodegeneration. J. Neurochem. 113, 363–
372.
Lieven C. J., Vrabec J. P. and Levin L. A. (2003) The effects of oxidative
stress on mitochondrial transmembrane potential in retinal ganglion
cells. Antioxid. Redox Signal. 5, 641–646.
Nguyen S. M., Alexejun C. N. and Levin L. A. (2003) Amplification of a
reactive oxygen species signal in axotomized retinal ganglion cells.
Antioxid. Redox Signal. 5, 629–634.
Okun Z., Kupershmidt L., Amit T., Mandel S., Bar-Am O., Youdim M.
B. and Gross Z. (2009) Manganese corroles prevent intracellular
nitration and subsequent death of insulin-producing cells. ACS
Chem. Biol. 4, 910–914.
Pacher P., Beckman J. S. and Liaudet L. (2007) Nitric oxide and per-
oxynitrite in health and disease. Physiol. Rev. 87, 315–424.
Robinson K. M., Janes M. S., Pehar M., Monette J. S., Ross M. F.,
Hagen T. M., Murphy M. P. and Beckman J. S. (2006) Selective
fluorescent imaging of superoxide in vivo using ethidium-based
probes. Proc. Natl Acad. Sci. USA 103, 15038–15043.
Saltsman I., Mahammed A., Goldberg I., Tkachenko E., Botoshansky M.
and Gross Z. (2002) Selective substitution of corroles: nitration,
hydroformylation, and chlorosulfonation. J. Am. Chem. Soc. 124,
7411–7420.
Lieven C. J., Schlieve C. R., Hoegger M. J. and Levin L. A. (2006)
Retinal ganglion cell axotomy induces an increase in intracel-
lular superoxide anion. Invest. Ophthalmol. Vis. Sci. 47, 1477–
1485.
Liu X., Mahammed A., Tripathy U., Gross Z. and Steer R. P. (2008)
Photophysics of Soret-excited tetrapyrroles in solution. III. Por-
phyrin analogues: aluminum and gallium corroles. Chem. Phys.
Lett. 459, 113–118.
Mackensen G. B., Patel M., Sheng H. et al. (2001) Neuroprotection from
delayed postischemic administration of a metalloporphyrin cata-
lytic antioxidant. J. Neurosci. 21, 4582–4592.
Mahammed A. and Gross Z. (2005) Albumin-conjugated corrole metal
complexes: extremely simple yet very efficient biomimetic oxida-
tion systems. J. Am. Chem. Soc. 127, 2883–2887.
Salvemini D., Wang Z. Q., Zweier J. L. et al. (1999) A nonpeptidyl
mimic of superoxide dismutase with therapeutic activity in rats.
Science 286, 304–306.
Schlieve C. R., Lieven C. J. and Levin L. A. (2006) Biochemical activity
of reactive oxygen species scavengers do not predict retinal gan-
glion cell survival. Invest. Ophthalmol. Vis. Sci. 47, 3878–3886.
Sheng H., Enghild J. J., Bowler R. et al. (2002) Effects of metal-
loporphyrin catalytic antioxidants in experimental brain ischemia.
Free Radic. Biol. Med. 33, 947–961.
Torreilles F., Salman-Tabcheh S., Guerin M. and Torreilles J. (1999)
Neurodegenerative disorders: the role of peroxynitrite. Brain Res.
Brain Res. Rev. 30, 153–163.
Zhao H., Kalivendi S., Zhang H., Joseph J., Nithipatikom K., Vasquez-
Vivar J. and Kalyanaraman B. (2003) Superoxide reacts with hy-
droethidine but forms a fluorescent product that is distinctly different
from ethidium: potential implications in intracellular fluorescence
detection of superoxide. Free Radic. Biol. Med. 34, 1359–1368.
Mahammed A. and Gross Z. (2006) Iron and manganese corroles are
potent catalysts for the decomposition of peroxynitrite. Angew
Chem. Int. Ed. Engl. 45, 6544–6547.
ꢀ 2010 The Authors
Journal Compilation ꢀ 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 488–498