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Sulforaphane Can Protect Lens Cells Against Oxidative Stress: Implications for Cataract Prevention

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Protecting the lens against oxidative stress is of great importance in delaying the onset of cataract. Isothiocyanates, such as sulforaphane (SFN), are proposed to provide cytoprotection against oxidative stress. We therefore tested the ability of sulforaphane to perform this role in lens cells and establish its ability to delay the onset of cataract. The human lens epithelial cell line FHL124 and whole porcine lens culture systems were used. The ApoToxGlo triplex assay was used to assess FHL124 cell survival, cytotoxicity and apoptosis. To determine single and double strand break DNA damage, the comet assay was performed and quantified. Lactate dehydrogenase levels in the medium were evaluated to reflect cell damage/death. To assess level of gene expression an Illumina whole genome HT-12 v4 beadchip was employed and protein expression validated by western blot and immunocytochemistry. 30μM H2O2 exposures to FHL124 cells caused a reduction in cell viability and increased cytotoxicity/apoptosis; these effects were significantly inhibited by 24h pre-treatment with 1μM SFN. In addition, 1μM SFN significantly reduced H2O2-induced DNA damage. When applied to cultured porcine lenses, SFN protected against H2O2-induced opacification. Illumina whole genome HT-12 v4 beadchip microarray data revealed 10 up-regulated genes following 24 hour exposure to 1μM SFN, which included NQO1 and TXNRD1. This pattern was confirmed at the protein level. The dietary component SFN demonstrates an ability to protect human lens cells against oxidative stress and thus could potentially delay the onset of cataract.
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Lens
Sulforaphane Can Protect Lens Cells Against Oxidative
Stress: Implications for Cataract Prevention
Hanruo Liu,
1
Andrew J. O. Smith,
1
Martin C. Lott,
1,2
Yongping Bao,
3
Richard P. Bowater,
1
John R. Reddan,
4
and Ian Michael Wormstone
1
1
School of Biological Sciences, University of East Anglia, Norwich, United Kingdom
2
School of Computing Sciences, University of East Anglia, Norwich, United Kingdom
3
Norwich Medical School, University of East Anglia, Norwich, United Kingdom
4
Oakland University, Rochester, Michigan
Correspondence: Ian Michael
Wormstone, School of Biological
Sciences, University of East Anglia,
Norwich Research Park, Norwich,
UK NR4 7TJ;
i.m.wormstone@uea.ac.uk.
Submitted: January 15, 2013
Accepted: June 16, 2013
Citation: Liu H, Smith AJO, Lott MC, et
al. Sulforaphane can protect lens cells
against oxidative stress: implications
for cataract prevention. Invest Oph-
thalmol Vis Sci. 2013;54:5236–5248.
DOI:10.1167/iovs.13-11664
PURPOSE. Protecting the lens against oxidative stress is of great importance in delaying the
onset of cataract. Isothiocyanates, such as sulforaphane (SFN), are proposed to provide
cytoprotection against oxidative stress. We therefore tested the ability of SFN to perform this
role in lens cells and establish its ability to delay the onset of cataract.
M
ETHODS. The human lens epithelial cell line FHL124 and whole porcine lens culture systems
were used. The ApoTox-Glo Triplex Assay was used to assess FHL124 cell survival,
cytotoxicity, and apoptosis. The MTS assay was used to assess cell populations. To determine
levels of DNA strand breaks, the alkaline comet assay was performed and quantified. Lactate
dehydrogenase levels in the medium were evaluated to reflect cell damage/death. To assess
level of gene expression, an Illumina whole-genome HT-12 v4 beadchip was used. Protein
expression was determined by Western blot and immunocytochemistry.
R
ESULTS. Exposures of 30 lMH
2
O
2
to FHL124 cells caused a reduction in cell viability and
increased cytotoxicity/apoptosis; these effects were significantly inhibited by 24-hour
pretreatment with 1 lM SFN. In addition, 1 lM SFN significantly reduced H
2
O
2
-induced
DNA strand breaks. When applied to cultured porcine lenses, SFN protected against H
2
O
2
-
induced opacification. Illumina whole-genome HT-12 v4 beadchip microarray data revealed
eight genes upregulated following 24-hour exposure to 1- and 2-lM SFN, which included
NQO1 and TXNRD1. This pattern was confirmed at the protein level. Nrf2 translocated to the
nucleus in response to 0.5- to 2.0-lM SFN exposure
C
ONCLUSIONS. The dietary component SFN demonstrates an ability to protect human lens cells
against oxidative stress and thus could potentially delay the onset of cataract.
Keywords: sulforaphane, Isothiocyanates, antioxidant, diet, lens, cataract
C
ataract renders millions in the world blind and is notably a
disease that largely afflicts the elderly.
1
In the world,
management of cataract is a significant strain on health care
budgets.
2
At present, the only means to treat cataract is by
surgical intervention
3
and it is predicted that 32 million
operations will be performed annually by 2020. Delaying the
onset of cataract is therefore a major health care priority across
the globe.
Free radical production leading to oxidative stress is an
initiating factor in the development of maturity-onset cata-
ract.
4
Free radicals are atomic or molecular species with at
least one unpaired electron in the outermost shell and any
free radical involving oxygen can be referred to as reactive
oxygen species (ROS). Unpaired electrons cause the free
radicals to be highly reactive and likely to take part in
chemical reactions.
5
ROS molecules include superoxide anion
(O
2
), hydroxyl radical (
OH), and hydrogen peroxide
(H
2
O
2
). Contrary to O
2
and
OH, which are extremely
unstable and react at or near their site of formation, H
2
O
2
is
less reactive, freely diffusible, and relatively long-lived.
5
ROS
can be generated either endogenously or exogenously in
relation to human cells. Endogenous sources include mito-
chondria, peroxisomes, lipoxygenases, nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase, and cytochrome
P450.
5
Exogenous sources include UV light, ionizing radiation,
chemotherapeutics, inflammatory cytokines, and environmen-
tal toxins.
6,7
It is widely believed that a rise in the intracellular
levels of ROS will damage various cell components, interrupt
physiological functions, and lead to aging and various
oxidative stress–associated disorders.
7
The damage can consist
of protein modification, lipid peroxidation, DNA base damage,
and fragmentation, all of which have been proposed to
contribute to cataractogenesis. The lens, like other organs, has
a well-designed defense system and uses primary defenses
against ROS to repair, recover, or degrade damaged mole-
cules.
8
The lens is able to defend itself against oxidation using
antioxidants from either enzymatic or nonenzymatic sys-
tems.
4,9
Primary antioxidants include nonenzymatic (e.g.,
glutathione, vitamin C, vitamin E, and carotenoids) and
enzymatic (e.g., superoxide dismutase, glutathione peroxi-
dase, and catalase) systems. The lens is known to contain
antioxidant defense systems. One example of a lens antiox-
idant defense system is the glutathione-dependent thioltrans-
ferase,
10–12
which may be critical in maintaining the lens in a
Copyright 2013 The Association for Research in Vision and Ophthalmology, Inc.
www.iovs.org j ISSN: 1552-5783
5236
reduced state by cleaving protein–thiol mixed disulfide bonds
formed upon the oxidation of lens proteins. Another example
is the NADPH-dependent thioredoxin/thioredoxin reductase
system,
13
which is very effective in reducing protein–protein
disulfide bonds and maintaining thiol/disulfide homeosta-
sis.
8,14
Under oxidative stress, some of these defense systems
can be upregulated.
15,16
A healthy lens uses its various
antioxidants and oxidation defense enzymes to maintain
crystallins, the structural proteins of the lens, in a reduced
state. This is necessary to maintain lens transparency
17
;
however, in the aging lens, protection and repair mechanisms
against oxidative stress slowly deteriorate or become ineffec-
tive and so the lens is less able to counteract the effects of
H
2
O
2
or other oxidants; thus, transparency is lost and cataract
can occur.
18
Therefore, enhancing the antioxidant defense
systems within the lens is a worthwhile aim and dietary
supplements provide a logical means to achieve this.
Isothiocyanates (ITCs), which are derived from glucosino-
lates found in cruciferous vegetables, are characterized by
sulfur-containing N ¼ C ¼ S functional groups. These include
allyl ITC from cabbage, mustard, and horseradish; benzyl
isothiocyanate; phenethyl ITC from watercress and garden
cress; and sulforaphane (SFN) from broccoli, cauliflower,
brassicas, and kale.
19
ITCs can inhibit many types of tumor
formation in animal models and their consumption is
inversely correlated with the risk of cancer in humans.
20
Protective mechanisms of ITCs have been proposed. Such
mechanisms include the induction of phase II detoxification
enzymes and inhibition of phase I carcinogen-activating
enzymes.
SFN is a product of hydrolytic conversion of 4-methysul-
phinylbutyl glucosinolate (glucoraphanin) by an endogenous
myrosinase.
21
It has been identified as a very potent
chemopreventive agent in numerous animal carcinogenesis
models as well as cell culture models, exerting its chemopre-
ventive effects through regulation of diverse molecular
mechanisms.
22
The most studied role of SFN in chemopreven-
tion is its ability to induce phase II detoxification enzymes as
well as cell cycle arrest and apoptosis. Experimental evidence
suggests that SFN activates NF-E2 p45–related factor-2 tran-
scription factor (Nrf2) in binding antioxidant response
elements in the promoter regions of target genes, thereby
increasing cellular defenses against oxidative stress.
22,23
It is
reported that most broccoli cultivars contain 2 to 10 lmol/g
glucosinolates.
20
If cooked, almost 100% of the glucosinolate is
converted to SFN. Intake of 200 lmol broccoli ITCs (mainly
SFN) in humans has been reported to result in SFN plasma
levels in the low micromolar range.
24
Therefore, the aim of this
current research was to assess the ability of SFN to protect
human lens epithelial cells against oxidative stress and lens
opacification.
METHODS
All reagents were purchased from Sigma (Poole, Dorset, UK)
unless otherwise stated.
Cell Culture
FHL124 is a nonvirally transformed cell line generated from
human capsule-epithelial explants,
25
showing a 99.5% homol-
ogy (in transcript profile) with the native lens epithelium.
26
FHL124 cells were routinely cultured at 358C in a humidified
atmosphere of 95% air and 5% CO
2
, in Eagle’s Minimum
Essential Medium (EMEM) supplemented with 5% vol/vol fetal
calf serum (Gibco, Paisley, UK) and 50 lg/mL gentamicin.
FHL124 cells were seeded on 35-mm tissue-culture dishes
(30,000/dish for Western blot, 30,000/dish for microarrays,
10,000/coverslip for immunocytochemistry, 35,000/dish for
alkaline comet assay), and 96-well plates (5000/well for
ApoTox-Glo Triplex Assay (Promega, Madison, WI), 10,000/
dish for lactate dehydrogenase [LDH] Assay).
ApoTox-Glo Triplex Assay
FHL124 cells were seeded on 96-well plates at a density of
5000 cells per well. Twenty-four hours before addition of
experimental conditions, culture medium was replaced with
200 lLserum-freeEMEM.Themediumwasthenremoved
from each well and replaced with fresh EMEM and test
compounds where appropriate. Plates were incubated at
358C, 5% CO
2
for the experimental duration (up to 72
hours). The ApoTox-Glo Triplex Assay (Promega) was used
to measure FHL124 cell viability, cytotoxicity, and apoptosis
following manufacturer’s instructions. Briefly, viability and
cytotoxicity are measured by uorescent signals produced
when either live-cell or dead-cell proteases cleave added
substrates GF-AFC (viability) and bis-AAF-R110 (cytotoxicity).
Fluorescence of the cleaved products is proportional to
either viability or cytotoxicity. GF-AFC can enter cells and is
therefore only cleavable by live-cell protease, which
incidentally becomes inactive when cell membrane activity
is lost; bis-AAF-R110 cannot enter the cell, and is cleaved
only by dead-cell protease leaked from cells lacking
membrane integrity. Both cleaved substrates have different
excitation and emission spectra. Apoptosis is measured by
the addition of a luminogenic caspase-3/7 substrate (Cas-
pase-Glo 3/7, a component of the ApoTox-Glo Triplex Assay;
Promega), which is cleaved in apoptotic cells to produce a
luminescent signal. Fluorescence was measured at 380
Ex
/
510
Em
(viability), 485
Ex
/520
Em
(cytotoxicity), and lumines-
cence (apoptosis) with a FLUOstar Omega plate reader
(BMG LabTech, Aylesbury, Bucks, UK).
MTS Assay
A cell proliferation assay (CellTiter 96 AQueous; Promega) was
used in accordance with the manufacturer’s instructions to
assess the viability of the cells. This assay is a colorimetric
method for determining the number of viable cells in
proliferation. The assay is based on the cellular conversion of
a tetrazolium salt (MTS) into a formazan product. The resultant
absorbance is directly proportional to the number of living cells
in culture. In brief, 5000 cells were seeded in 96-well plates for
24 hours before the medium was replaced with 200 lL serum-
free EMEM and incubated for a further 24 hours. The medium
was then removed from each well and replaced with 200 lL
fresh EMEM; test compounds were added where appropriate.
Cells were maintained in experimental conditions for 24 or 48
hours. Then, 25 lL CellTiter 96 AQueous One Solution was
added directly to the culture wells and incubated for the final
hour. Absorbance was measured at 490 nm with a spectropho-
tometric plate reader (FLUOstar Omega plate reader; BMG
LabTech). Cell viability was expressed as a percentage, with
100% representing the signal from untreated cells and 0%
representing the background signal from empty wells.
Cell Death Assay (LDH Assay)
A nonradioactive cytotoxicity assay (Cyto Tox 96R; Roche,
Welwyn Garden City, UK) was used to measure the release of
LDH from the cultured human lens cells and porcine whole
lens cultures. The procedure followed the manufacturer’s
protocol. The plate was read at 490 nm with a FLUOstar
Omega plate reader (BMG LabTech).
Sulforaphane Can Protect the Lens IOVS j August 2013 j Vol. 54 j No. 8 j 5237
Alkaline Comet Assay
The alkaline comet assay, also called single-cell gel electropho-
resis, is a sensitive and rapid technique for quantifying and
analyzing DNA strand breaks in individual cells. FHL124 cells
were seeded onto 35-mm plastic culture dishes at a density of
35,000 cells per dish and grown until approximately 70%
confluent. At this time point, the medium was removed from
each dish and replaced with 1.5 mL serum-free EMEM for 24
hours before placing the cells in experimental conditions for a
further 24 hours. Cells were pretreated with 1 lM SFN for 24
hours before exposure to 30 lMH
2
O
2
and cells incubated at
358C, 5% CO
2
. The cells were washed with ice-cold PBS,
harvested, counted, resuspended in PBS containing 10%
dimethyl sulfoxide and frozen at 808C until the alkaline comet
assay was performed. Samples were defrosted and approxi-
mately 25,000 cells per sample were centrifuged at 108g for 5
minutes at 48C. Pellets were resuspended in 0.6% low melting
point agarose, dispensed in duplicate onto glass microscope
slides (precoated in 1% normal melting point agarose), and
allowed to set on ice, under a glass coverslip. Once set, the
coverslips were removed and slides transferred into ice-cold
lysis buffer (100 mM disodium EDTA [Fisher Scientific, Lough-
borough, UK], 2.5 M NaCl [Fisher Scientific], 10 mM Tris-HCl
[Formedium; Fisher Scientific], pH 10.0 with 1% Triton X-100
added immediately before use) for 1 hour. Slides were washed
twice with ice-cold dH
2
O for 10 minutes, transferred to a
atbed electrophoresis tank, and incubated in freshly prepared
ice-cold electrophoresis buffer (300 mM NaOH [BDH Merck
Ltd., Poole, Dorset, UK], 1 mM disodium EDTA, pH 13) for 30
minutes, followed by electrophoresis in the same buffer at 21 V
(1 V/cm) for 30 minutes. Procedures were performed protected
from direct light. Slides were drained of electrophoresis buffer
and flooded with neutralization buffer (0.4 M Tris-HCl, pH 7.5)
for 30 minutes, washed twice in dH
2
O for 10 minutes, and dried
at 378C. Slides were stained with SYBR Green I nucleic acid
stain diluted from a 10,000 X stock in 1X TE buffer (10 mM Tris-
HCl, 1 mM EDTA) for 5 minutes protected from light at room
temperature, drained, and dried at room temperature before
visualization. For each sample, 100 comets were randomly
analyzed (50 per gel), with images captured by uorescence
microscopy (Axioplan 2; Zeiss, Cambridge, UK) and comets
scored using Comet Assay IV Lite analysis software (Perceptive
Instruments, Bury St Edmunds, UK).
Microarray Analysis
FHL124 cells were seeded onto 35-mm plastic culture dishes at a
density of 35,000 cells per dish and grown until approximately
70% confluent. At this time point, the medium was removed
from each dish and replaced with 1.5 mL serum-free EMEM for
24 hours before placing the cells in experimental conditions for
a further 24 hours. One dish was used per experimental
condition; the experiment was carried out on four separate
occasions. A microarray platform was selected to provide a
comprehensive view of the gene activity under the different
conditions. To achieve this, the commercially available Human-
HT12.v4 Expression Bead Chip (BD-25-113; Illumina, San Diego,
CA) array platform was used. RNA was extracted from FHL124
cells using an RNeasy mini kit (Qiagen Ltd., Crawley, UK). The
monolayer of cells was lysed by adding 350 lLbufferRLT
containing 2-b-mercaptoethanol, to each culture dish. The cell
lysate was collected using a cell scraper. The cell lysate was then
transferred into a sterile microcentrifuge tube and was passed
through a 20-gauge needle (0.9-nm diameter), fitted to an RNase-
free syringe, three times. This process homogenizes the cells
allowing the RNA to be released from all FHL124 cells; 350 lL
70% ethanol was added to the homogenized lysate, and was
mixed via pipetting. The ethanol was added to the lysate to
create conditions that promote selective binding of RNA to the
RNeasy silica-gel membrane. Then, 700 lL of the solution was
transferred to an RNeasy mini column placed in a 2-mL
collection tube. The columns were centrifuged for 30 seconds
at 6000g and the ow-through discarded. A series of washes
using different buffers was carried out to produce the pure RNA.
Then, 700 lL RW1 buffer was added to the RNeasy mini column,
centrifuged for 30 seconds, and removal of ow-through was
carried out. The RNeasy mini column was fitted to one new
collection tube and washed two times with 500 lL RPE buffer
with an initial centrifugation for 15 seconds and the second
centrifugation for 2 minutes to dry the RNeasy silica-gel
membrane containing the RNA. Using 50 lL RNeasy free water,
the RNA was eluted form the column into a sterile 1.5-mL
microcentrifuge tube and centrifuged for 1 minute. RNA samples
were shipped on dry ice to a commercial microarray facility. RNA
quantity and quality was assessed using the NanoDrop ND-1000
spectrophotometer (NanoDrop Technologies, Wilmington, DE)
and an Agilent RNA pico labchip (Wokingham, UK); only
samples with an RNA integrity number greater than or equal to 9
were used in the study. Average RNA yields per dish were 879 6
248 ng, 1003 6 231 ng, and 975 6 211 ng for the 0-, 1-, and 2-lM
SFN-treated groups respectively. From all samples, 100 ng RNA
was processed according to the Illumina Whole-Genome Gene
Expression Direct Hybridisation Assay Guide, using the Ambion
Kit: Illumina TotalPrep-96 RNA Amplification Kit. Qualitative and
quantitative quality control was performed on the labeled cRNA
and 1.5 lg labeled cRNA was hybridized to a HumanHT-12.v4
Beadchip and scanned by the Illumina BeadArray reader.
Illumina microarray (BeadArray) unnormalized probe pro-
file data were analyzed using the Bioconductor package
(provided in the public domain at http://www.bioconductor.
org) in R (provided in the public domain at http://www.
r-project.org). First, the data from different chips were loaded
into R to be background corrected, quantile normalized, and
variance stabilized.
27
The normalized data from all the arrays
have been deposited in the Array Express database with an
accession number (E-MEXP-3923). Lists of differentially ex-
pressed genes were computed by using the eBAYES statistic
28
to compute a P value. Additionally, the fold change (FC) of each
gene was computed, that is, the ratio of average expression
level between the two groups. Differentially expressed genes
were determined by using a combination of FC and adjusted P
value (q value) threshold criteria, as that has been found to
discover genes more likely to play a physiological role.
29
The
Benjamini and Hochberg method
30
is used to compute q values
and control the false discovery rate, that is, the proportion of
genes identified as being significant but that later transpire as
being false leads. Genes were identified as being significant if
FC was greater than or equal to 1.3 and q value was greater
than or equal to 0.20.
Western Blot Analysis
Cell lysates from FHL124 cells were prepared using Daub’s lysis
buffer supplemented with 1 mM phenylmethylsulfonyl fluoride
(PMSF) and 10 lg/mL aprotinin for 20 minutes on ice and
centrifuged at 16,060g for 10 minutes. The protein content
was determined by the BCA assay (Bio-Rad, Hemel Hempsted,
UK) so that equal amounts of protein per sample were loaded
onto 8% SDS-polyacrylamide gels and transferred to PVDF
membrane using a semidry transfer cell. The membrane was
blocked with PBS containing 5% wt/vol nonfat dry milk and
0.1% vol/vol Tween-20, hybridized with primary antibody
followed by incubation with secondary antibody (Amersham
Biosciences, Bucks, UK). Proteins were detected using the ECL
plus blotting analysis system (Amersham Biosciences).
Sulforaphane Can Protect the Lens IOVS j August 2013 j Vol. 54 j No. 8 j 5238
Immunocytochemistry
FHL124 cells were grown on sterile glass coverslips contained
within a 35-mm plastic culture dish at a density of 10,000 cells
per coverslip and treated with 0-, 0.5-, 1-, and 2-lM SFN for 24
hours. Cells were fixed with 4% formaldehyde in PBS for 30
minutes and permeabilized with PBS containing 0.5% Triton X-
100 for 30 minutes. Three washes were made in PBS-BSA-Igepal
(0.02% wt/vol and 0.05% vol/vol, respectively). Nonspecific sites
were blocked with normal goat or donkey serum (1:50 in 1% wt/
vol BSA in PBS). NQO1 antibody and TXNRD1 antibody were
diluted 1:200 and Nrf2 antibody was diluted 1:100 in 1% BSA in
PBS and applied overnight at 48C followed by washing three
times for 5 minutes with shaking with 0.02% BSA, 0.05% IGEPAL
in PBS. NQO1 and TXNRD1 were visualized using ALEXA 488-
conjugated goat anti-mouse secondary antibody and nrf2 using
ALEXA 488-conjugated donkey anti-rabbit secondary antibody
diluted 1:100 in 1% BSA in PBS (Molecular Probes, Leiden, The
Netherlands). Chromatin was stained with 4’,6-Diamidino-2-
Phenylindole, Dihydrochloride (DAPI) to reveal nuclei (1:100 in
FIGURE 1. Concentration-dependent effects of SFN on FHL124 cell viability (A, D), cytotoxicity (B, E), and apoptosis (C, F) detected by the
ApoTox-Glo Triplex Assay following a 24- (AC) and 72-hour (DF) culture period with SFN. The data are presented as mean 6 SEM (n ¼4). The
asterisk indicates a significant difference between the treated group and untreated controls (P 0.05; ANOVA with Dunnett’s post hoc test).
Sulforaphane Can Protect the Lens IOVS j August 2013 j Vol. 54 j No. 8 j 5239
1% wt/vol BSA in PBS) (Molecular Probes). The stained
preparations were again washed extensively and mounted on
microscope slides with Hydromount mounting medium (Na-
tional Diagnostics, Hull, UK). Images were viewed using
uorescence microscopy (Axioplan 2; Zeiss), and applicable
images were quantified using ImageJ1.45s analysis software
(available in the public domain at http://rsbweb.nih.gov/ij/).
31
FIGURE 2. The viability of FHL124 cells exposed to 30 lMH
2
O
2
together with 1 lM SFN over a 24-hour culture period using the MTS
assay. The data are expressed as % cell survival in comparison with
control, represented as 100%. Each column represents the mean 6
SEM of four independent experiments. The asterisk represents a
significant difference between the indicated groups (P 0.05; ANOVA
with Tukey’s post hoc test).
FIGURE 3. The viability of FHL124 cells exposed to 1 lM SFN for 24
hours, followed by 30 lMH
2
O
2
in the absence of SFN (A) and in the
presence of SFN (B) for a further 24 hours using the MTS assay. The data
are expressed as % cell survival in comparison with control, represented
as 100%. Each column represents the mean 6 SEM of four independent
experiments. The asterisk represents a significant difference between
indicated groups (P 0.05; ANOVA with Tukey’s post hoc test).
FIGURE 4. SFN protection against oxidative stress induced loss of cell
viability (A), cytotoxicity (B), and apoptosis (C), following a 24-hour
experimental period, determined using the ApoTox-Glo Assay. Cells
were pretreated with 1 lM SFN for 24 hours before exposure to 30 lM
H
2
O
2
. Data are presented as mean 6 SEM (n ¼ 4). The asterisk
indicates a significant difference between the indicated groups (P
0.05; ANOVA with Tukey’s post hoc test).
FIGURE 5. SFN protection against oxidative stress–induced cell
damage/death, following a 24-hour experimental period, determined
using the LDH assay. Cells were pretreated with 1 lM SFN for 24 hours
before exposure to 30 lMH
2
O
2
. Data are expressed as mean 6 SEM (n
¼ 4). The asterisk represents a significant difference between the
indicated groups (P 0.05; ANOVA with Tukey’s post hoc test).
Sulforaphane Can Protect the Lens IOVS j August 2013 j Vol. 54 j No. 8 j 5240
Whole Pig Lens Culture
Fresh porcine eyes were obtained from a local slaughterhouse
(Felthorpe, Norfolk, UK). The tissue collection conformed to
the ARVO Statement for the Use of Animals in Ophthalmic and
Vision Research. Eyes were placed in sterile containers and
covered with Eagle’s minimum essential medium (EMEM)
containing 200 U/mL penicillin and 200 lg/mL streptomycin.
They were stored at 48C before dissection. Within 24 hours
postmortem, lenses were dissected by anterior approach
following cornea removal and incubated in 3 mL bicarbonate-
FIGURE 6. SFN suppression of DNA damage of FHL124 cells induced by oxidative stress detected using the comet assay following a 30-, 60-, 120-, or
240-minute experimental period. Cells were pretreated with 1 lM SFN for 24 hours before exposure to 30 lMH
2
O
2
. DNA strand breaks were
measured by the alkaline comet assay and tails were measured for at least 100 comets per sample. Data are presented as mean 6 SEM pooled from
four individual experiments. The asterisk represents a significant difference between the indicated groups (P 0.05; ANOVA with Tukey’s post hoc
test).
FIGURE 7. SFN reduces hydrogen peroxide–induced lens opacity. (A) Representative bright-field and dark-field images of whole pig lens organ
cultures over time. (B) Pooled data showing LDH levels within the culture medium at end point; data are presented as mean 6 SEM (n ¼ 4). (C)
Quantification of lens opacity over time (n¼4); data are presented as mean 6 SEM (n ¼4). SFN was applied at 2 lM and H
2
O
2
at 2 mM. The asterisk
represents a significant difference from all other groups (P 0.05; ANOVA with Tukey’s post hoc test).
Sulforaphane Can Protect the Lens IOVS j August 2013 j Vol. 54 j No. 8 j 5241
CO
2
-buffered EMEM (pH 7.4), containing 100 U/mL penicillin,
100 lg/mL streptomycin, 0.25 lg/mL amphotericin, and 50 lg/
mL gentamicin at 358C. After a preculture period of 24 to 72
hours to ensure no damage had arisen from the isolation
procedure, lenses were exposed to 3 mL EMEM or 3 mL EMEM
supplemented with SFN(2 lM) for 24 hours before addition of
H
2
O
2
(2 mM final concentration) or vehicle control. During the
experimental period, lens images were taken at the starting
point (t ¼ 0), 24-hour time point, and 4-day point using a
charge-coupled device (CCD) camera (UVP, Cambridge, UK)
with Synoptics software (GeneSnap; Synoptics, Cambridge,
UK). At the end of 24 hours of culture in the presence of
experimental conditions, the medium was collected to assay
for LDH. Dark-field images of lenses taken using the CCD
camera were measured for grayscale values with ImageJ1.45s
analysis software (available in the public domain at http://
rsbweb.nih.gov/ij/). Images captured at the start of the
experimental period (t ¼ 0 days) were treated as background
level for each lens. Subsequent grayscale values of images for
each lens, captured at day 1 and day 4, were background
corrected.
Statistical Analyses
A t-test analysis was performed using Excel software (Micro-
soft, Redmond, WA) to determine any statistical difference
between the two groups. One-way ANOVA with Tukey’s post
hoc analysis was used to assess multiple groups when all or
many pairwise comparisons were of interest. One-way ANOVA
with Dunnett’s post hoc analysis was used to assess all groups
compared against one control group. A 95% confidence
interval was used to assess significance.
RESULTS
Sulforaphane Effects on Cell Viability,
Cytotoxicity, and Apoptotic Cell Death
Cell viability relative to the untreated control group was not
significantly affected by SFN exposure of 5 lM and below
following 24- and 72-hour exposure periods (Figs. 1A, 1D).
However, at SFN concentrations of 10 lM and above, a
significant reduction in cell viability was observed; this effect
became more pronounced with increasing concentrations of
SFN (Figs. 1A, 1D). A significant increase in cytotoxicity was
also seen with SFN exposure between 20 and 100 lM (Figs. 1B,
1E). Apoptosis was detected in the ApoTox-Glo Triplex Assay
(Promega) using Caspase-Glo to detect Caspase 3/7 activity
and, consistent with the other measurements, a significant
increase was identified following 10- to 100-lM SFN exposure
(Figs. 1C, 1F).
Sulforaphane Protection of Lens Cells Against
Oxidative Stress
SFN concentrations of 5 lM and less did not reduce viability of
FHL124 cells and, thus, these concentrations could be used to
assess putative protection of lens cells against oxidative insult
(by hydrogen peroxide).
TABLE 1. SFN (1 lM) Induced Gene Expression Increase Detected in FHL124 Epithelial Cells Using Illumina Gene Microarray
Official
Symbol Official Full Name Location Summary FC P Value q Value
NQO1 NAD(P)H dehydrogenase,
quinone 1
16q22.1 This protein’s enzymatic activity prevents the 1-
electron reduction of quinones that results in
the production of radical species
2.04735 1.51E-05 0.087413526
OKL38 Oxidative stress–induced
growth inhibitor
16q23.3 Encodes an oxidative stress response protein
that regulates cell death
1.96899 3.04E-07 0.010537
LOC392437 Unknown Unknown Unknown 1.63879 0.000023 0.113791741
TXNRD1 Thioredoxin reductase 1 12q23-q24.1 This gene encodes a member of the family of
pyridine nucleotide oxidoreductases and plays
a role in selenium metabolism and protection
against oxidative stress
1.60769 1.41E-05 0.087413526
PIR pirin (iron-binding nuclear
protein)
XP22.2 The encoded protein is an Fe(II)-containing
nuclear protein expressed in all tissues
1.41567 4.59E-06 0.053056757
It can act as a transcriptional cofactor and is
involved in the regulation of DNA
transcription and replication
G6PD Glucose-6-phosphate
dehydrogenase
xq28 This gene encodes glucose-6-phosphate
dehydrogenase whose main function is to
produce NADPH
1.39306 2.8E-06 0.048589621
EPHX1 Epoxide hydrolase 1,
microsomal (xenobiotic)
1q42.1 Epoxide hydrolase is a critical biotransformation
enzyme, which functions in both the
activation and detoxification of epoxides
1.37074 1.17E-05 0.087413526
FTL Ferritin, light polypeptide 19q13.33 FTL encodes the light subunit of the ferritin
protein
1.35383 5.39E-05 0.169818382
It affects the rates of iron uptake and release in
different tissues
PANX2 Pannexin 2 22Q13.33 This protein and pannexin 1 are abundantly
expressed in the central nervous system and
are coexpressed in various neuronal
populations
1.32656 2.69E-05 0.116426773
All genes presented were increased 1.3-fold in the SFN-treated group relative to nontreated control and were statistically different (q 0.20;
eBAYES t-test with Benjamini-Hochberg correction).
Sulforaphane Can Protect the Lens IOVS j August 2013 j Vol. 54 j No. 8 j 5242
FHL124 cells were cotreated with 1 lM SFN and 30 lM
H
2
O
2
for 24 hours and cell viability tested by the MTS assay
(Fig. 2). The 30 lMH
2
O
2
reduced FHL124 cell viability to 55%
of the control population; 1 lM SFN alone had no effect on cell
viability, which did not significantly differ from the control
group (Fig. 2). Cells cotreated with 1 lM SFN and 30 lMH
2
O
2
exhibited a reduction in the viable cell population, which was
significantly different from the control group, but did not
significantly differ from the cells treated with 30 lMH
2
O
2
alone (Fig. 2), indicating no significant protection of the cells
by SFN against H
2
O
2
toxicity using this approach.
To investigate whether SFN has indirect antioxidant
properties that could elicit protection to lens cells against
oxidative stress, FHL124 cells were incubated with 1 lM SFN
for 24 hours before exposure to 30 lMH
2
O
2
. SFN was then
removed (Fig. 3A) or retained (Fig. 3B) before adding H
2
O
2
.
Figure 3B shows that addition of 30 lMH
2
O
2
significantly
reduced the viable cell population (again detected using the
MTS assay), within 24 hours, such that levels were 56% 6 4%
compared with the untreated control. FHL124 cells pretreated
with 1 lM SFN, before addition of 30 lMH
2
O
2
and retained for
the experimental duration, demonstrated a significant increase
in the viable population relative to H
2
O
2
alone that was
comparable with control populations. When FHL124 cells
were incubated with SFN for 24 hours before its removal and
subsequent H
2
O
2
addition, marked cytoprotection was still
observed (Fig. 3A).
Due to the results of the MTS assay, the ApoTox-Glo Triplex
Assay (Promega) was used so as to have an independent means
of verifying the results. Exposure of FHL124 cells to 30 lM
H
2
O
2
for a 24-hour period resulted in a significant decrease in
cell viability and a significant increase in cytotoxicity and
apoptosis (Fig. 2). In agreement with the above experiments,
addition of 1 lM SFN to the cells had no discernible effect on
cell viability, cytotoxicity, or apoptosis relative to serum-free
maintained control cells (Fig. 4). However, pretreatment of
cells with 1 lM SFN significantly inhibited H
2
O
2
-induced
effects, such that cell viability, cytotoxicity, and apoptosis did
not significantly differ from serum-free or SFN (alone)
maintained cells (Fig. 4).
To support the ApoTox-Glo Triplex Assay (Promega) data,
the LDH assay was used to assess cell damage/death. Treatment
with 30 lM hydrogen peroxide invoked a significant increase
in LDH release into the medium (Fig. 5). This effect was
inhibited by pretreatment of the cells with 1 lM SFN. No
difference in LDH levels was observed in the SFN-alone group
compared with serum-free controls (Fig. 5).
Numerous prior studies have identified that oxidative stress
induces DNA strand breaks in human cells.
32,33
To investigate
such effects in this experimental system, the alkaline comet
assay was used to investigate DNA strand breaks (and their
repair) induced by oxidative stress in FHL124 cells over time.
Exposure to 30 lMH
2
O
2
resulted in the greatest levels of DNA
strand breaks in cells harvested at the 30-minute time point,
which demonstrated a mean value for DNA in the tail of 52.7%
(Fig. 6). This declined with time, but remained significantly
elevated at the 2-hour time point. There were significantly
lower levels of DNA strand breaks if cells were pretreated with
1 lM SFN (Fig. 6), indicating an enhanced antioxidant defense.
Treatment with 1 lM SFN alone for this time period did not
significantly change levels of DNA strand breaks when
compared with untreated cells.
To further test the protective nature of SFN against
oxidative stress, we used a porcine whole-lens culture
TABLE 2. SFN (2 lM) Induced Gene Expression Increase Detected in FHL124 Epithelial Cells Using Illumina Gene Microarray
Official
Symbol Official Full Name Location Summary FC P Value q Value
OKL38 Oxidative stress–induced
growth inhibitor
16q23.3 Encodes an oxidative stress response protein
that regulates cell death
2.18195 5.51E-08 0.001911616
NQO1 NAD(P)H dehydrogenase,
quinone 1
16q22.1 This protein’s enzymatic activity prevents the 1-
electron reduction of quinones that results in
the production of radical species
2.09728 1.06E-05 0.046724545
LOC392437 Unknown Unknown Unknown 1.74835 6.19E-06 0.046724545
TXNRD1 Thioredoxin reductase 1 12q23-q24.1 This gene encodes a member of the family of
pyridine nucleotide oxidoreductases and plays
a role in selenium metabolism and protection
against oxidative stress
1.72358 3.18E-06 0.036786402
PIR Pirin (iron-binding nuclear
protein)
XP22.2 The encoded protein is an Fe(II)-containing
nuclear protein expressed in all tissues
1.56149 2.63E-07 0.004559489
It can act as a transcriptional cofactor and is
involved in the regulation of DNA
transcription and replication
GCLM Glutamate-cysteine ligase,
modifier subunit
1p22.1 Glutamate-cysteine ligase is the first rate limiting
enzyme of glutathione synthesis
1.53588 5.97E-05 0.159341311
FTL Ferritin, light polypeptide 19q13.33 FTL encodes the light subunit of the ferritin
protein
1.42567 1.06E-05 0.046724545
It affects the rates of iron uptake and release in
different tissues
EPHX1 Epoxide hydrolase 1,
microsomal (xenobiotic)
1q42.1 Epoxide hydrolase is a critical biotransformation
enzyme, which functions in both the
activation and detoxification of epoxides
1.37391 1.08E-05 0.046724545
G6PD Glucose-6-phosphate
dehydrogenase
xq28 This gene encodes glucose-6-phosphate
dehydrogenase. Its main function is to
produce NADPH
1.34738 0.000009 0.046724545
All genes presented were increased 1.3-fold in the SFN-treated group relative to nontreated control and were statistically different (q 0.20;
eBAYES t-test with Benjamini-Hochberg correction).
Sulforaphane Can Protect the Lens IOVS j August 2013 j Vol. 54 j No. 8 j 5243
system.
34
Following dissection, whole porcine lenses did not
demonstrate any notable opacity. Lenses maintained in serum-
free medium remained transparent over the 4-day culture
period. This is shown in Figure 7A, as the grid placed beneath
the lens can be clearly seen. Similarly, with the dark-field image
presented in Figure 7, minimal white light scattering regions
are observed. Addition of 2 lM SFN to the cultures did not
affect transparency, such that lenses appeared similar to the
serum-free control group. Exposure to 2 mM H
2
O
2
induced a
marked change in transparency that appeared as a cloudiness
in the peripheral cortex that progressed over time, such that
most of the cortical region was affected (Fig. 7A). At day 1, the
peripheral lens had begun to opacify, such that the grid could
not be seen clearly through these regions (Fig. 7A) and was
associated with light scatter (Fig. 7A). When 2 mM H
2
O
2
was
added in the presence of 2 lM SFN, opacity was still observed
but this was less marked than the H
2
O
2
-only treated group. At
end point (day 4), the culture medium was analyzed for LDH
(Fig. 7B). Serum-free and SFN-only treated lenses had no
discernible levels of LDH in the culture medium. Addition of
hydrogen peroxide induced a dramatic increase in LDH levels,
which was reduced in the presence of SFN.
Identification of SFN Protective Mechanisms
As an initial screen to identify gene changes induced by SFN, an
Illumina gene microarray was performed. The data revealed
that nine genes were significantly upregulated following a 24-
hour exposure to 1 lM SFN (Table 1) and nine genes were also
upregulated by 2 lM SFN (Table 2). There were eight genes
upregulated in both 1- and 2-lM SFN exposures respectively
(Fig. 8) (Table 1). NQO1 and TXNRD1 are classic phase II
enzymes and were thus assessed using Western blot and
immunocytochemistry to detect the protein products. In
accordance with the microarray data, levels of NQO1 and
TXNRD1 were significantly elevated in response to SFN. A
concentration-dependent increase in NQO1 and TXNRD1
protein level was observed using Western blot methods (Fig.
9). Significant elevation was observed at SFN concentrations
greater than or equal to 0.5 lM, with a peak observed with 2
lM SFN. This was a 2.5-fold elevation relative to untreated
control cells for TXNRD1 (Fig. 9B). Similarly, NQO1 protein
levels were increased by 2.3-fold compared with untreated
control cells after 24-hour incubation with 2 lM SFN (Fig. 9B).
Protein expression was further evaluated using immunocyto-
chemistry (Fig. 10). Using this approach, the distribution of
both NQO1 and TXNRD1 was largely cytoplasmic. Quantifica-
tion of these images also demonstrated a significant increase in
protein levels, such that with 2 lM SFN, an 8-fold and 13-fold
increase relative to untreated control cells was observed for
NQO1 and TXNRD1, respectively (Fig. 10B).
SFN Induction of Nrf2 Signaling in FHL124 Cells
As Nrf2 is a likely candidate in the regulation of SFN protection,
further investigation was carried out to determine whether
SFN could induce Nrf2 nuclear translocation in human lens
epithelial cells. The accumulation and translocation of Nrf2 to
the nucleus would indicate activation of Nrf2 and the
induction of the Keap1-Nrf2-ARE pathway. Immunocytochem-
istry experiments were carried out to identify the location of
Nrf2 proteins within FHL124 cells following SFN treatment for
4 hours. As shown in Figure 11A, only cytoplasmic labeling of
Nrf2 with no distinct nuclear staining was observed in the
nonstimulated cells, whereas an intense nuclear labeling was
observed in the SFN-stimulated cells. In addition, the SFN
treatment of the cells led to a dose-dependent increase in
nuclear Nrf2 levels (Fig. 11B). These data clearly show that
Nrf2 is translocated to the nucleus, indicating Keap1-Nrf2-ARE
pathway activation in response to SFN exposure.
FIGURE 8. A Venn diagram showing the effect of 24-hour exposure of
1- and 2-lM SFN on increased gene expression of FHL124 cells
identified by Illumina gene microarray.
FIGURE 9. SFN induced NQO1 and TXNRD1 protein increase in
FHL124 cells, detected using Western blot. (A) Representative blots
showing NQO1, TXNRD1, and b-actin levels within FHL124 cells. (B)
Quantitative data derived from band intensities; the protein band
intensities for NQO1 and TXNRD1 were normalized to b-actin. Data are
presented as mean 6 SEM (n ¼ 4). The asterisk indicates a significant
difference between the treated and the nontreated control group (P
0.05; ANOVA with Dunnett’s post hoc test).
Sulforaphane Can Protect the Lens IOVS j August 2013 j Vol. 54 j No. 8 j 5244
DISCUSSION
Cataract is the major cause of blindness worldwide and
cataract surgery is the most common surgical procedure
performed on the elderly.
3
The large cost of this operation and
possible complications associated with it favor long-term goals
of avoiding cataract formation, or significantly retarding onset
of the disease.
Epidemiological evidence suggests that high fruit and
vegetable intake is associated with lower risk of any form of
cataracts, although the mechanisms of protection are not
known.
35
SFN has been identified in numerous cell and animal
carcinogenesis models to be an effective chemopreventive
agent, which uses a diverse range of molecular mechanisms to
achieve this.
20
The current work demonstrates that pretreat-
ment with SFN, which is abundant in green vegetables, such as
broccoli, yielded protection for lens epithelial cells against
hydrogen peroxide–induced DNA damage, cell death, and
transparency loss. It is therefore of interest to consider the
mechanisms by which SFN provides this protection.
19
Putative
mechanisms include a direct interaction with the oxidative
stressor (H
2
O
2
), inhibition of apoptotic signals, or via
modification of DNA repair systems enhancing activity of
antioxidant defense enzymes.
36
From the experimental data, it is unlikely that SFN causes its
effect by direct antioxidant actions because with cotreatment
no significant protection was observed; to achieve protection,
pretreatment was required. ITCs can rapidly accumulate in
human and animal cells, with the peak intracellular ITC
accumulation reached within 0.5 to 3.0 hours of exposure and
up to 100- to 200-fold over the extracellular ITC concentra-
tion.
37,38
This suggests modification to the cell occurs to
enhance protection or that transport of SFN to key areas within
the cells takes time. It is clear SFN can be readily accumulated
in cells, but it is unclear how modification of SFN itself could
influence the beneficial outcomes observed. SFN reacts with
glutathione to give rise to a glutathione-SFN conjugate, which
is catalyzed by Glutathione-S-Transferases.
38
After that, step-
wise cleavage of glutamine and glycine first by the enzymes c-
glutamyl transpeptidase and then by cysteinylglycinase yields
an L-cysteine conjugate. Then N-acetyltransferase acetylates
the L-cysteine conjugate to produce N-acetyl-L-cysteine conju-
gate (mercapturic acid derivative). Understanding the molec-
ular life cycle of SFN and its derivates in lens cells over time
will be of great interest and may provide further understanding
FIGURE 10. Immunocytochemical analyses of NQO1 and TXNRD1 distribution and expression within FHL124 cells following exposure to SFN for a
24-hour period. (A) Representative images showing the organization of NQO1 and TXNRD1 within FHL124 cells. (B) Quantitative data derived from
uorescence micrographs showing changes in protein level in response to SFN. Data are presented as mean 6 SEM (n ¼4). The asterisk indicates a
significant difference between treated and control groups (P 0.05; ANOVA with Dunnett’s post hoc test).
Sulforaphane Can Protect the Lens IOVS j August 2013 j Vol. 54 j No. 8 j 5245
of how SFN can be regulated to provide maximum benefit to
an individual. Establishing a greater understanding of when
pretreatment of SFN is effective and the point at which peak
levels are observed in both cultured cells and within the lens
will be of great interest for future studies.
DNA strand breaks detected using the alkaline comet assay
were reduced by SFN pretreatment. This may be a result of
upstream effects of SFN giving rise to less damage or because
of the increased efficiency of mechanisms that repair DNA
strand breaks, such as nonhomologous end joining.
36
This can
be tested in the future by the use of small interfering RNA
(siRNA) knockdowns of the repair systems. If upstream factors
reduce DNA damage, then no difference will be observed by
reduction of DNA repair capacity.
Modification of enzymatic antioxidant defense systems is
also likely to play a key role and the data presented support
this notion. Dietary ITCs are breakdown products of glucosi-
nolates from cruciferous vegetables, for example, broccoli.
20
There are many studies that demonstrate ITCs are potent
inducers of chemopreventive enzymes, including antioxidant
enzymes.
39,40
The current study has addressed this issue by an
Illumina gene array to determine the effects of SFN on gene
expression. The data revealed that eight genes were signifi-
cantly upregulated following 24-hour exposure to both 1- and
2-lM SFN, of which a number are associated with antioxidant
defense. Of the genes that were upregulated, all are reported in
the literature to be controlled by the transcription factor Nrf2.
TXNRD1 and NQO1 are classic phase II enzymes and have
been reported to show increased expression in a number of
cells and tissues.
41
G6PD encodes glucose-6-phosphate dehy-
drogenase, which is a cytosolic enzyme whose primary
function is to produce NADPH; this gene is also reported to
FIGURE 11. SFN induction of nuclear translocation of Nrf2 in FHL124 cells. (A) Representative uorescent micrographs showing Nrf2 (green)
distribution following a 4-hour exposure to 0-, 0.5-, 1-, and 2-lM SFN treatment. Nuclei and actin filaments are labeled using DAPI (blue) and Texas
red X-Phalloidin (red), respectively. (B) Quantitative data derived from uorescence micrographs showing changes in nuclear protein level in
response to SFN. Data are presented as mean 6 SEM (n ¼4). The asterisk indicates a significant difference between treated and control groups (P
0.05; ANOVA with Dunnett’s post hoc test).
Sulforaphane Can Protect the Lens IOVS j August 2013 j Vol. 54 j No. 8 j 5246
be regulated by Nrf2 signaling.
42
Moreover, NADPH is used by
NQO1 as a hydride donor in the conversion of quinones to
hydroquinones. Therefore, upregulation of G6PD is likely to
facilitate the antioxidant activity of NQO1. Nrf2 signaling has
also been implicated in the expression of pirin in small airway
epithelium. In smokers, Nrf2 signaling was more active and
this was associated with increased expression of pirin relative
to nonsmokers.
43
Moreover, it was identified that the promoter
region of the pirin gene contains functional antioxidant
response elements.
43
In addition, EPHX1 and FTL, which
encode epoxide hydrolase 1 and ferritin light chain, respec-
tively, were also shown in a mouse model to be induced by 1-
(2-cyano-3-,12-dioxooleana-1,9[11]-dien-28-oyl)imidazole
(CDDO-Im), which is a highly potent chemopreventive agent.
In Nrf2 knockout animals, the induction of EPHX1 was
suppressed indicating an important role of Nrf2 signaling in
its regulation.
44
In the case of FTL, the CCDO-Im induced
expression was ablated in the absence of Nrf2.
45
OKL38,
which encodes oxidative stress–induced growth inhibitor 1, is
reported to show induction following oxidative stress.
46
In
cancer cells, it is believed that following DNA strand breaks,
OKL38 interacts with p53 and relocates to the mitochondrion
to initiate cytochrome c release and apoptosis; OKL38 is
therefore defined as a tumor suppressor.
47
In the current
system, OKL38 is upregulated in response to SFN, but under
these conditions, cells continue to survive and grow. In
addition, cytotoxicity/apoptosis does not differ from controls.
Oxidative stress–induced DNA damage is suppressed by SFN
and perhaps without this cue, OKL38 does not interact with
p53 and induce apoptosis. The expression of OKL38 remains
curious and further inhibition studies will be required to
elucidate its putative role in SFN protection in the lens.
The common factor linking all the genes shown to be
upregulated is Nrf2/keap1 signaling. It was therefore of
importance to establish whether Nrf2 signaling can take place
in lens cells in response to SFN. The data confirm that Nrf2
translocation does occur and thus it is reasonable to
hypothesize that the protection observed with SFN against
oxidative stress is largely mediated by Nrf2 regulation. SFN is
known to influence this pathway through interaction with the
thiol group on keap1, which liberates Nrf2 from the complex.
Nrf2 then translocates to the nucleus and initiates transcrip-
tion.
20
It will therefore be of great interest in the future to
determine more of the role of Nrf2 in expression of the genes
identified from the microarray data. Such work will involve
establishing the kinetics of Nrf2 nuclear translocation in
response to SFN using immunocytochemistry and GFP tags,
Nrf2/ARE reporter assays, and siRNA approaches to assess
functional involvement of the individual genes and Nrf2
signaling.
In summary, pretreatment of cells or whole lenses with SFN
can modify antioxidant defense mechanisms by induction of
the Keap1-Nrf2-ARE pathway, thus rendering lens cells more
capable of suppressing the daily insult of oxidative stress.
Improved intake through an SFN-rich diet or use of supple-
ments could provide a novel approach to retard the onset of
cataract formation in the human lens.
Acknowledgments
The authors thank Sarah Russell for cell culture assistance and Julie
Eldred for technical advice.
Supported by The Humane Research Trust.
Disclosure: H. Liu, None; A.J.O. Smith, None; M.C. Lott, None;
Y. Bao, None; R.P. Bowater, None; J.R. Reddan, None; I.M.
Wormstone, None
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Sulforaphane Can Protect the Lens IOVS j August 2013 j Vol. 54 j No. 8 j 5248
... LECs are well equipped with antioxidant systems to combat oxidative stress [7]. When the ROS exceeds the antioxidant defensive protection in the LECs, it will result in oxidative damage which leads to apoptosis of LECs and cataract formation [4,8]. At present, the only means to treat cataract is by surgical intervention. ...
... All data were expressed as means ± SD. * P < 0:05 vs. the control group; # P < 0:05 vs. the IR group. 8 Oxidative Medicine and Cellular Longevity Nrf2 signal pathway is highly associated with oxidative stress in the lens [13,42]. Nrf2 has been proved to be maintained at a low level through 26S proteasome-mediated degradation and Keap1-mediated ubiquitylation. ...
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Ionizing radiation- (IR-) induced oxidative stress has been recognized as an important mediator of apoptosis in lens epithelial cells (LECs) and also plays an important role in the pathogenesis of IR-induced cataract. Ferulic acid (FA), a phenolic phytochemical found in many traditional Chinese medicine, has potent radioprotective and antioxidative properties via activating nuclear factor erythroid 2-related factor 2 (Nrf2) signal pathway. The goals of this study were to determine the protective effect of FA against IR-induced oxidative damage on human lens epithelial cells (HLECs) and to elucidate the role of Nrf2 signal pathway. HLECs were subjected to 4 Gy X-ray radiation with or without pretreatment of FA. It was found that FA pretreatment protected HLECs against IR-induced cell apoptosis and reduced levels of ROS and MDA caused by radiation in a dose-dependent manner. IR-dependent attenuated activities of antioxidant enzymes (SOD, CAT, and GPx) and decreased ratio of reduced GSH/GSSG were increased by pretreatment of FA. FA inhibited IR-induced increase of Bax and cleaved caspase-3 and the decrease of Bcl-2 in a dose-dependent manner. Furthermore, FA provoked Nrf2 nuclear translocation and upregulated mRNA and protein expressions of HO-1 in a dose-dependent manner. These findings indicated that FA could effectively protect HLECs against IR-induced apoptosis by activating Nrf2 signal pathway to inhibit oxidative stress, which suggested that FA might have a therapeutic potential in the prevention and alleviation of IR-induced cataract.
... Hydrogen peroxide (H 2 O 2 ) is a non-free radical derived from the ROS family that can easily penetrate the lipid membrane and produce toxicity in the lens (11). Previous studies have shown that ROS production is stimulated by H 2 O 2 -induced epithelial cell injury and protein degradation, similar to human cataract damage (12). Therefore, H 2 O 2 has been widely used to induce apoptosis of HLECs in order to simulate cataract formation in cell models (13). ...
... Therefore, the study of cataracts should focus on the exploration of new therapeutic targets. Apoptosis of lens epithelial cells is universally acknowledged to be closely related to the formation of cataracts, and OS induced by ROS has been considered as a major contributor to apoptosis of lens epithelial cells (12,24). In this study, H 2 O 2 was used to construct a cell model of oxidative damage in HLECs. ...
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Background: Long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) plays a regulatory role in many biological processes; however, its role in cataracts has yet to be illuminated. This study aimed to investigate the protective role of NEAT1 in hydrogen peroxide (H2O2)-treated human lens epithelial cells (HLECs) and its underlying molecular mechanism. Methods: HLECs (SRA01/04) were treated with 300 µM H2O2 to mimic cataract in vitro. Cell viability was detected by performing an MTT assay and EdU staining. Flow cytometry was carried out to detect apoptosis of HLECs. DNA damage was examined using γ-H2A histone family member X staining. and reactive oxygen species (ROS) production was measured using 2',7'dichlorofluorescin diacetate staining. The expression levels of lncRNA and proteins were detected with quantitative real-time polymerase chain reaction and western blot, respectively. Results: The expression of NEAT1 was observed to be increased in H2O2-treated HLECs and age-related cataract (ARC) tissues. Knockdown NEAT1 strongly protected against H2O2-induced cell death and also regulated the expression of cleaved caspase-3, B-cell lymphoma 2, and Bcl-2-associated X protein. Further, knockdown NEAT1 also significantly suppressed H2O2-induced intracellular ROS production and malondialdehyde (MDA) content, but elevated the glutathione (GSH) activity of H2O2-treated cells. Also, it is demonstrated that si-NEAT1 greatly inhibited H2O2-induced phosphorylation of NF-кB p65 and p38 MAPK. Conclusions: This study confirmed that knockdown NEAT1 attenuated H2O2-induced damage in HLECs, and inhibited the oxidative stress and apoptosis of HLECs via regulating nuclear factor-kappa B (NF-κB) p65 and p38 MAPK signaling. It may provide a potential target for clinical treatment of cataracts.
... [22] Liu et al. suggested that sulforaphane, a sulfur-rish compound in cruciferous vegetables, could protect human lens cells against oxidative stress based on an in-vitro study. [27] Most previous studies demonstrated an inverse association between higher intake of carotene and lutein, which are abundant in green and yellow vegetables, and reduced risk of cataract. [28][29] However, both the BMES and JPHC study, as well as the current study, found no signi cant association between cataract and green or yellow vegetable intake. ...
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Purpose: To investigate the association between fruit and vegetable (F&V) intakeand the risk of cataract. Design: Prospective cohort study. Methods: We included 72,160 participants who were free of cataract at baseline from the UK Biobank. Frequency and type of F&V intake were assessed using a web-based 24-h dietary questionnaire from 2009 to 2012. Development of cataract during the follow-up was defined by self-report or hospital inpatient records up to 2021. Cox proportional regression models were used to estimate the association between F&V intake and incident cataract. Results: During a mean follow-up of 9.1 years, 5753 participants developed cataract with a corresponding incidence of 8.0%. After adjusting for multiple demographic, medical and lifestyle covariates, higher intake of F&V were associated with a lower risk of cataract (≥6.5 vs. <2 servings/week: hazards ratio [HR]: 0.82, 95% CI: 0.76 to 0.89; P < 0.0001). Regarding specific types, significant reduced risk of cataract was found for higher intake of legumes (P = 0.0016), tomatoes (≥5.2 vs. <1.8 servings/week: HR: 0.94, 95% CI: 0.88 to 1.00), and apple and pear (>7 vs. <3.5 servings/week: 0.89, 95% CI: 0.83 to 0.94; P < 0.0001), but not for cruciferous vegetables, green leafy vegetables, berry, citrus fruit or melon. Smokers were found to benefit more from F&V intake than former and never smokers. Men also could benefit more from higher vegetable intake than women. Conclusions: More F&V intake, especially legumes, tomatoes, apple and pear was associated with lower risk of cataract in this UK Biobank cohort.
... 4 This hormetic chemical displays contrasting properties depending on concentration and induces myriad cellular events. 5,6 At low doses, SFN can trigger the activation of phase-II detoxification enzymes, increase antioxidant defense, decrease inflammatory responses, and promote cell survival via the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. High levels of SFN block cell proliferation and induce cell death. ...
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Article
Purpose: Sulforaphane (SFN) is a therapeutic phytochemical agent for many health conditions. SFN-induced cytotoxicity is shown to have promise in preventing posterior capsule opacification (PCO). In the current study, we aimed to elucidate key processes and mechanisms linking SFN treatment to lens cell death. Methods: The human lens epithelial cell line FHL124 and central anterior epithelium were used as experimental models. Cell death was assessed by microscopic observation and cell damage/viability assays. Gene or protein levels were assessed by TaqMan RT-PCR or immunoblotting. Mitochondrial networks and DNA damage were assessed by immunofluorescence. Mitochondrial membrane potential, activating transcription factor 6 (ATF6) activity, ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), and glutathione reductase (GR) activity were measured using different light reporter assays. SFN metabolites were analyzed by LC-MS/MS. Results: Treatment with N-acetylcysteine (NAC), a reactive oxygen species scavenger, prevented SFN-induced cell death in both models. NAC also significantly protected FHL124 cells from SFN-induced mitochondrial dysfunctions, endoplasmic reticulum stress (ERS), DNA damage and autophagy. SFN significantly depleted GSH, the major antioxidant in the eye, and reduced GR activity, despite doubling its protein levels. The most abundant SFN conjugate detected in lens cells following SFN application was SFN-GSH. The addition of GSH protected lens cells from all SFN-induced cellular events. Conclusions: SFN depletes GSH levels in lens cells through conjugation and inhibition of GR activity. This leads to increased reactive oxygen species and oxidative stress that trigger mitochondrial dysfunction, ERS, autophagy, and DNA damage, leading to cell death. In summary, the work presented provides a mechanistic understanding to support the therapeutic application of SFN for PCO and other disorders.
... A healthy lens maintains the structural proteins of the lens in a reduced state by its defense system. However, in the aging lens, protection and repair mechanisms slowly deteriorate or become ineffective to counteract the oxidant [155]. Advanced glycation end products (AGEs) formation due to oxidant reactions dramatically accelerated cataractogenesis [156]. ...
Article
The aging process deteriorates organs' function at different levels, causing its progressive decline to resist stress, damage, and disease. In addition to alterations in metabolic control and gene expression, the rate of aging has been connected with the generation of high amounts of Reactive Oxygen Species (ROS). The essential perspective in free radical biology is that reactive oxygen species (ROS) and free radicals are toxic, mostly cause direct biological damage to targets, and are thus a major cause of oxidative stress. Different enzymatic and non-enzymatic compounds in the cells have roles in neutralizing this toxicity. Oxidative damage in aging is mostly high in particular molecular targets, such as mitochondrial DNA and aconitase, and oxidative stress in mitochondria can cause tissue aging across intrinsic apoptosis. Mitochondria's function and morphology are impaired through aging, following a decrease in the membrane potential by an increase in peroxide generation and size of the organelles. Telomeres may be the significant trigger of replicative senescence. Oxidative stress accelerates telomere loss, whereas antioxidants slow it down. Oxidative stress is a crucial modulator of telomere shortening, and that telomere-driven replicative senescence is mainly a stress response. The age-linked mitochondrial DNA mutation and protein dysfunction aggregate in some organs like the brain and skeletal muscle, thus contributing considerably to these post-mitotic tissues' aging. The aging process is mostly due to accumulated damage done by harmful species in some macromolecules such proteins, DNA, and lipids. The degradation of non-functional, oxidized proteins is a crucial part of the antioxidant defenses of cells, in which the clearance of these proteins occurs through autophagy in the cells, which is known as mitophagy for mitochondria.
... Morin (3, 5, 7, 20, 40-pentahydroxyflavone), widely used in herbal medicines, has been shown to increase the Nrf2 protein levels and stimulate the extracellular signal-regulated kinase (ERK)-Nrf2 signaling pathway in human LECs, leading to the upregulation of HO-1 and Nrf2 cytoprotective effects against oxidative stress [62]. Plant-extracted isothiocyanate 1-isothiocyanato-4-methyl-sulfinyl butane (SFN) has gained attention as a potential nutritional anti-cataract therapy by its ability to increase the activity of thioredoxin reductase in the lens of mouse, which prevents oxidative stress and cataract formation when consumed [63]. The multi-target neuroprotective drug, DL-3-n-butylphthalide (NBP), is widely utilized to treat ischemic stroke patients and diminishes oxidative damage, enhances the function of the mitochondria, lessens inflammation, and decreases neuronal apoptosis [64]. ...
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Cataracts account for over half of global blindness. Cataracts formations occur mainly due to aging and to the direct insults of oxidative stress and inflammation to the eye lens. The nuclear factor-erythroid-2-related factor 2 (Nrf2), a transcriptional factor for cell cytoprotection, is known as the master regulator of redox homeostasis. Nrf2 regulates nearly 600 genes involved in cellular protection against contributing factors of oxidative stress, including aging, disease, and inflammation. Nrf2 was reported to disrupt the oxidative stress that activates Nuclear factor-κB (NFκB) and proinflammatory cytokines. One of these cytokines is matrix metalloproteinase 9 (MMP-9), which participates in the decomposition of lens epithelial cells (LECs) extracellular matrix and has been correlated with cataract development. Thus, during inflammatory processes, MMP production may be attenuated by the Nrf2 pathway or by the Nrf2 inhibition of NFκB pathway activation. Moreover, plant-based polyphenols have garnered attention due to their presumed safety and efficacy, nutritional, and antioxidant effects. Polyphenol compounds can activate Nrf2 and inhibit MMP-9. Therefore, this review focuses on discussing Nrf2's role in oxidative stress and cataract formation, epigenetic effect in Nrf2 activity, and the association between Nrf2 and MMP-9 in cataract development. Moreover, we describe the protective role of flavonoids in cataract formation, targeting Nrf2 activation and MMP-9 synthesis inhibition as potential molecular targets in preventing cataracts.
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Age-related cataract (ARC) is one of the leading blinding eye diseases worldwide. Chronic oxidative stress and the apoptosis of human lens epithelial cells (HLECs) have been suggested to be the mechanism underlying cataract formation. Acetyl-11-keto-β-boswellic acid (AKBA) is a pentacyclic triterpene with antioxidative and antiapoptotic effects. In this study, we investigated the potential effects of AKBA on oxidative-induced HLECs injury and cataract formation. H2O2 was used to simulate HLECs oxidative injury in vitro, and Na2SeO3 was applied to establish an in vivo cataract model. In our current study, a cell counting kit-8 (CCK-8) assay was performed to evaluate the effects of H2O2 and AKBA on cell viability in vitro. Intracellular reactive oxygen species (ROS) levels were measured with the ROS assay to verify the antioxidant capacity of AKBA. Apoptotic cells were detected and measured by TUNEL staining and flow cytometry, and quantitative real-time (qRT)-PCR and Western blotting were applied to examine the transcription and expression of apoptosis-related proteins. Furthermore, immunofluorescence staining was performed to locate factor-erythroid 2-related factor 2 (Nrf2), and the protein levels of Nrf2, kelch-like ECH-associated protein 1 (Keap1) and heme oxygenase-1 (HO-1) were determined by Western blotting. Finally, we observed the degree of lens opacity and performed hematoxylin-eosin (H&E) staining to assess the protective effect of AKBA on cataract formation in vivo. AKBA increased HLECs viability under H2O2 stimulation, decreased intracellular ROS levels and alleviated the cell apoptosis rate in vitro. AKBA significantly decreased the expression of caspase-3 and Bax and increased the content of Bcl-2. The results of immunofluorescence and immunohistochemical staining proved that the expression and nuclear translocation of Nrf2 were activated with AKBA treatment in vivo and in vitro. Moreover, computational docking results showed that AKBA could bind specifically to the predicted Keap1/Nrf2 binding sites. After AKBA activation, Nrf2 dissociates from the Nrf2/Keap1 complex, translocates into the nucleus, and subsequently promotes HO-1 expression. In addition, AKBA attenuated lens opacity in selenite-induced cataracts. Overall, these findings indicated that AKBA alleviated oxidative injury and cataract formation by activating the Keap1/Nrf2/HO-1 cascade. Therefore, our current study highlights that AKBA may serve as a promising treatment for ARC progression.
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Purpose: Age-related cataracts affect the majority of older adults and are a leading cause of blindness worldwide. Treatments that delay cataract onset or severity have the potential to delay cataract surgery, but require relevant animal models that recapitulate the major types of cataracts for their development. Unfortunately, few such models are available. Here, we report the lens phenotypes of aged mice lacking the critical antioxidant transcription factor Nfe2l2 (designated as Nrf2 -/-). Methods: Three independent cohorts of Nrf2 -/- and wild-type C57BL/6J mice were evaluated for cataracts using combinations of slit lamp imaging, photography of freshly dissected lenses, and histology. Mice were fed high glycemic diets, low glycemic diets, regular chow ad libitum, or regular chow with 30% caloric restriction. Results: Nrf2 -/- mice developed significant opacities between 11 and 15 months and developed advanced cortical, posterior subcapsular, anterior subcapsular, and nuclear cataracts. Cataracts occurred similarly in male mice fed high or low glycemic diets, and were also observed in 21-month male and female Nrf2 -/- mice fed ad libitum or 30% caloric restriction. Histological observation of 18-month cataractous lenses revealed significant disruption to fiber cell architecture and the retention of nuclei throughout the cortical region of the lens. However, fiber cell denucleation and initiation of lens differentiation was normal at birth, with the first abnormalities observed at 3 months. Conclusions: Nrf2 -/- mice offer a tool to understand how defective antioxidant signaling causes multiple forms of cataract and may be useful for screening drugs to prevent or delay cataractogenesis in susceptible adults.
Article
Purpose: To investigate the potential function of Ginsenoside Rg1 (Rg1) against lens opacification. Methods: Eyeballs from adult Sprague-Dawley rats were enucleated and lenses were dissected for ex vivo culture under H2O2 treatment. Water soluble protein (WSP) content, the level of superoxide dismutase (SOD), total glutathione (GSH), and reduced GSH were detected by indicated assays. Cell viability was performed by Cell Counting Kit-8 experiment. Results: Exposure of 0.2 mM H2O2 in lenses resulted in obvious cloudiness and typical pathological changes of cataract such as rupture of the lens capsule, degenerative lens epithelial cells (LECs), etc. Rg1 effectively prevented lens opacity caused by H2O2. After Rg1 treatment, lens WSP content, the level of SOD, total GSH, and reduced GSH were increased, while the level of MDA and oxidized GSH were decreased. In addition, MDA concentration of lens by Rg1 treatment only was found to be lower than the controls. Rg1 attenuated H2O2-induced cell injury at the concentration of 0.4 mM that it elevated cell activity, and peaked at 0.6 mM. Conclusions: This study demonstrated that Rg1 might have the capability to protect lens against oxidative stress-induced cataract, at least by local administration. Abbreviations: LECs: lens epithelial cells; Rg1: Ginsenoside Rg1; SD: Sprague-Dawley; ROS: reactive oxygen species; SOD: Superoxide Dismutase; GSH: glutathione; MDA: Malonediadehyde; H2O2: Hydrogen peroxide
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Viral corneal infection is a common cause of visual impairment and blindness. Polyinosinic‑polycytidylic acid, or poly(I:C), is similar to viral double‑stranded RNA in structure and has been implicated in the release of a variety of cytokines, chemokines and matrix metalloproteinases (MMPs) by corneal fibroblasts. Sulforaphane (SFN) is an isothiocyanate compound found in cruciferous vegetables. The present study investigated the potential effect of SFN on the poly(I:C)‑stimulated release of cytokines, chemokines and MMPs in human corneal fibroblasts (HCFs). ELISA showed that SFN was associated with a time‑ and dose‑dependent reduction in poly(I:C)‑stimulated production of interleukin (IL)‑8, chemoattractant protein‑1, IL‑6, MMP‑1 and MMP‑3 by HCFs. Western blot analysis indicated that SFN suppressed the function of poly(I:C) by modulating mitogen‑activated protein kinases (MAPKs), including p38 and extracellular signal‑regulated kinase (ERK), activator protein‑1 (AP‑1) component c‑Jun and the kinase, Akt, and the phosphorylation and degradation of the nuclear factor (NF)‑κB inhibitor IκB‑α. Immunofluorescence analysis revealed that SFN attenuated the production of poly(I:C)‑induced nuclear translocation of the NF‑κB p65 subunit. Reverse transcription‑quantitative PCR analysis revealed that SFN prevented the poly(I:C)‑induced upregulation of Toll‑like receptor 3 (TLR3) mRNA expression in HCFs. No significant cytotoxic effect of SFN on HCFs was observed. In summary, SFN attenuated the poly(I:C)‑induced production of proinflammatory chemokines, cytokines and MMPs by HCFs, by inhibiting TLR3, MAPK (p38 and ERK), AP‑1, Akt and NF‑κB signaling. SFN may therefore be a potential novel treatment for viral corneal infection by limiting immune cell infiltration.
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The transcription factor Nrf2 is responsible for regulating a battery of antioxidant and cellular protective genes, primarily in response to oxidative stress. A member of the cap 'n' collar family of transcription factors, Nrf2 activation is tightly controlled by a series of signaling events. These events can be separated into the basal state, a preinduction response, gene induction, and finally a postinduction response, culminating in the restoration of redox homeostasis. However, despite the immensely intricate level of control the cellular environment imposes on Nrf2 activity, there are many opportunities for perturbations to arise in the signaling events that favor carcinogenesis and, therefore, implicate Nrf2 as both a tumor suppressor and a protooncogene. Herein, we highlight the ways in which Nrf2 is regulated, and discuss some of the Nrf2-inducible antioxidant (NQO1, NQO2, HO-1, GCLC), antiapoptotic (Bcl-2), metabolic (G6PD, TKT, PPARγ), and drug efflux transporter (ABCG2, MRP3, MRP4) genes. In addition, we focus on how Nrf2 functions as a tumor suppressor under normal conditions and how its ability to detoxify the cellular environment makes it an attractive target for other oncogenes either via stabilization or degradation of the transcription factor. Finally, we discuss some of the ways in which Nrf2 is being considered as a therapeutic target for cancer treatment.-Shelton, P., Jaiswal, A. K. The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene?
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The tumor suppressor p53 is a well-known transcription factor controlling the expression of its target genes involved in cell cycle and apoptosis. In addition, p53 also plays a direct proapoptotic role in mitochondria by regulating cytochrome c release. Recently, we identified a novel downstream target of p53, OKL38, which relocalizes from nucleus to mitochondria upon forced expression to induce apoptosis. However, the mechanism underlying OKL38 targeting to mitochondria and apoptosis induction remains unclear. Here, we found that OKL38 interacts with p53 to regulate mitochondria function. After DNA damage, OKL38 colocalizes with p53 to mitochondria in U2OS cells. Further, p53 and OKL38 are targeted to mitochondria in synergy: forced expression of OKL38 leads to p53 localization to mitochondria while the expression of a mitochondria enriched p53 polymorphic variant, p53(R72), leads to OKL38 enrichment in mitochondria. Biochemical analyses found that OKL38 and p53 interact in vivo and in vitro via multiple domains. In cell biological assays, multiple regions of OKL38 mediate its mitochondria localization and induce mitochondria morphology changes. OKL38 induces formation of megamitochondria and increases cellular levels of reactive oxygen species. Furthermore, OKL38 induces cytochrome c release upon incubation with mitochondria. Taken together, our studies suggest that OKL38 regulates mitochondria morphology and functions during apoptosis together with p53.
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For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the analysis of scientific images. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.
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PurposeTo investigate relationships between a wide range of macro- and micronutrients, including antioxidant vitamins, and the three main types of cataract in older people.
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Studies in mono-culture of cells have shown that diesel exhaust particles (DEPs) increase the production of reactive oxygen species (ROS) and oxidative stress-related damage to DNA. However, the level of particle-generated genotoxicity may depend on interplay between different cell types, e.g. lung epithelium and immune cells. Macrophages have important immune defence functions by engulfing insoluble foreign materials, including particles, although they might also promote or enhance inflammation. We investigated the effect of co-culturing type II lung epithelial A549 cells with macrophages upon treatment with standard reference DEPs, SRM2975 and SRM1650b. The exposure to DEPs did not affect the colony-forming ability of A549 cells in co-culture with THP-1a cells. The DEPs generated DNA strand breaks and oxidatively damaged DNA, measured using the alkaline comet assay as formamidopyrimidine-DNA glycosylase or oxoguanine DNA glycosylase (hOGG1) sensitive sites, in mono-cultures of A549 or THP-1a and co-cultures of A549 and THP-1a cells. The strongest genotoxic effects were observed in A549 mono-cultures and SRM2975 was more potent than SRM1650b. The ROS production only increased in cells exposed to SRM2975, with strongest concentration-dependent effect in the THP-1a mono-cultures. The basal respiration level in THP-1a cells increased on exposure to SRM1650b and SRM2975 without indication of mitochondrial dysfunction. This is consistent with activation of the cells and there was no direct relationship between levels of respiration and ROS production. In conclusion, exposure of mono-cultured cells to DEPs generated oxidative stress to DNA, whereas co-cultures with macrophages had lower levels of oxidatively damaged DNA than A549 epithelial cells.
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Ochratoxin A is one of the most abundant food-contaminating mycotoxins worldwide, and its immunosuppressive effects in human caused more and more concern in biomedical field. In the present study, the toxicity of OTA on human peripheral blood mononuclear cells (hPBMC) was explored by analyzing the involvement of oxidative pathway. It was found that OTA treatment led to the release of reactive oxygen species (ROS) and the increase of 8-hydroxydeoxyguanosine (8-OHdG), an important biomarker of oxidative DNA stress. Moreover, we found that OTA treatment induced DNA strand breaks in hPBMC as evidenced by DNA comet tails formation and increased γ-H2AX expression. In addition, OTA could induce cell cycle arrest at G1 phase by down-regulating the expression of CDK4 and cyclinD1 protein, as well as apoptosis in hPBMC in vitro. Pre-treatment of hPBMC with antioxidant, N-acetyl-L-cysteine (NAC), could reduce OTA-induced ROS release and DNA damage, thus confirming the involvement of oxidative DNA damage in the OTA genotoxicity in hPBMC. NAC pre-treatment could also significantly prevent OTA-induced down-regulation of CDK4 and cyclinD1 expression in hPBMC. All the results demonstrated the involvement of oxidative pathway in OTA mediated cytotoxicity in human immune cells, which including the ROS accumulation-oxidative DNA damage-G1 arrest and apoptosis. Our results provide new insights into the molecular mechanisms by which OTA might promote immunotoxicity.
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Protein-thiol mixed disulfide formation has been implicated as a possible mechanism for the protein-protein aggregation in cataractogenesis. Previously we have found that two species of thiols are bound to proteins: GSH (PSSG) and cysteine (PSSC). In this study we found these molecules are ubiquitous in animal lenses with the highest levels in human, dog and rat, and lowest in monkey. However, the relative amount of PSSG to PSSC is quite different in each animal species. The ratio of was in rat lens, in human and dog lenses and in monkey lens. We also studied the effect of aging on the protein-thiol mixed disulfide levels in human donor lenses between 3 months and 88 years. Lens GSH levels were inversely related to age, similar to earlier reports, but PSSC levels increased linearly with age. PSSG levels showed a triphasic pattern with an initial sharp and linear increase from a low content in infants to a highest level at age 20; fell back about 50% to a new steady state level that was maintained for four more decades; finally, above 60 years, the levels in some lenses were two to three-fold higher while some lenses remained at the same low value. PSSC in human lens appeared to concentrate in the nuclear region and in the water insoluble proteins while PSSG was more evenly distributed. Besides the aging effect on the protein-thiol mixed disulfides, oxidative stress also potentiated protein modification in the human lens. As demonstrated by the hydrogen peroxide induced cataract in organ culture, old human lenses showed biochemical and morphological changes similar to those in published rat lens studies under the same conditions except that human lenses were more resistant to oxidation and in addition to PSSG, the PSSC level was also elevated.