S-allylcysteine scavenges singlet oxygen and hypochlorous acid and protects LLC-PK1 cells of potassium dichromate-induced toxicity

Article abstract of DOI:10.1016/j.fct.2007.05.002

It has been found that S-allylcysteine (SAC), a garlic-derived compound, has in vivo and in vitro antioxidant properties. In addition, it is known that SAC is able to scavenge different reactive oxygen or nitrogen species including superoxide anion (O₂^{{radical dot} -}), hydrogen peroxide (H₂O₂), hydroxyl radical (OH^{{radical dot}}), and peroxynitrite anion (ONOO^-) although the IC_5O values for each reactive species has not been calculated and the potential ability of SAC to scavenge singlet oxygen (¹O₂) and hypochlorous acid (HOCl) has not been explored. The purposes of this work was (a) to explore the potential ability of SAC to scavenge ¹O₂ and HOCl, (b) to further characterize the O₂^{{radical dot} -}, H₂O₂, OH^{{radical dot}}, and ONOO^- scavenging ability of SAC by measuring the IC₅₀ values using in vitro assays, and (c) to explore the potential ability of SAC to ameliorate the potassium dichromate (K₂Cr₂O₇)-induced cytotoxicity in LLC-PK1 cells in which oxidative stress is involved. The scavenging activity was compared against the following reference compounds: N-acetylcysteine for O₂^{{radical dot} -}, sodium pyruvate for H₂O₂, dimethylthiourea for OH^{{radical dot}}, lipoic acid and glutathione for ¹O₂, lipoic acid for HOCl, and penicillamine for ONOO^-. It was found that SAC was able to scavenge concentration-dependently all the species assayed with the following IC_5O (mean ± SEM, mM): O₂^{{radical dot} -} (14.49 ± 1.67), H₂O₂ (68 ± 1.92), OH^{{radical dot}} (0.68 ± 0.06), ¹O₂ (1.93 ± 0.27), HOCl (2.86 ± 0.15), and ONOO^- (0.80 ± 0.05). When the ability of SAC to scavenge these species was compared to those of the reference compounds it was found that the efficacy of SAC (a) to scavenge O₂^{{radical dot} -}, H₂O₂, OH^{{radical dot}}, and ONOO^- was lower, (b) to scavenge HOCl was similar, and (c) to scavenge ¹O₂ was higher. In addition, it was found that SAC was able to prevent K₂Cr₂O₇-induced toxicity in LLC-PK1 cells in culture. It was showed for the first time that SAC is able to scavenge ¹O₂ and HOCl and to ameliorate the K₂Cr₂O₇-induced toxicity.

Full text of DOI:10.1016/j.fct.2007.05.002

Food and Chemical Toxicology 45 (2007) 2030–2039
www.elsevier.com/locate/foodchemtox
S-allylcysteine scavenges singlet oxygen and hypochlorous acid
and protects LLC-PK cells of potassium dichromate-induced toxicity
1
a
b
a
a
Omar Noel Medina-Campos , Diana Barrera , Sabina Segoviano-Murillo , Diana Rocha ,
c
b
a,
*
Perla D. Maldonado , Nicandro Mendoza-Pati n˜ o , Jos e´ Pedraza-Chaverri
a
Facultad de Qu ´ı mica, Departamento de Biolog ´ı a, Edificio F, Sequndo Piso, Laboratorio 209, Universidad Nacional Aut o´ noma de M e´ xico (UNAM),
Ciudad Universitaria, 04510, D.F., Mexico
Facultad de Medicina, Departamento de Farmacolog ´ı a, Universidad Nacional Aut o´ noma de M e´ xico (UNAM), Ciudad Universitaria, 04510, D.F., Mexico
b
c
Laboratorio de Patolog ´ı a Vascular Cerebral, Instituto Nacional de Neurolog ´ı a y Neurocirug ´ı a ‘‘Manuel Velasco Su a´ rez’’, M e´ xico D.F. 14269, Mexico
Received 24 October 2006; accepted 7 May 2007
Abstract
It has been found that S-allylcysteine (SAC), a garlic-derived compound, has in vivo and in vitro antioxidant properties. In addition, it
Åꢀ
is known that SAC is able to scavenge different reactive oxygen or nitrogen species including superoxide anion (O ), hydrogen peroxide
2
Å
ꢀ
H O ), hydroxyl radical (OH ), and peroxynitrite anion (ONOO ) although the IC values for each reactive species has not been
2 2 5O
(
1
calculated and the potential ability of SAC to scavenge singlet oxygen ( O
The purposes of this work was (a) to explore the potential ability of SAC to scavenge O
2
) and hypochlorous acid (HOCl) has not been explored.
and HOCl, (b) to further characterize the
O , H O , OH , and ONOO scavenging ability of SAC by measuring the IC50 values using in vitro assays, and (c) to explore the poten-
1
2
Åꢀ
Å
ꢀ
2
2
2
tial ability of SAC to ameliorate the potassium dichromate (K Cr O )-induced cytotoxicity in LLC-PK1 cells in which oxidative stress is
2
2
7
Åꢀ
involved. The scavenging activity was compared against the following reference compounds: N-acetylcysteine for O , sodium pyruvate
2 2 2
for H O , dimethylthiourea for OH , lipoic acid and glutathione for O , lipoic acid for HOCl, and penicillamine for ONOO . It was
2
Å
1
ꢀ
found that SAC was able to scavenge concentration-dependently all the species assayed with the following IC (mean ± SEM, mM):
5
O
Åꢀ
Å
1
ꢀ
O
(14.49 ± 1.67), H O (68 ± 1.92), OH (0.68 ± 0.06), O (1.93 ± 0.27), HOCl (2.86 ± 0.15), and ONOO (0.80 ± 0.05). When the
2
2
2
2
ability of SAC to scavenge these species was compared to those of the reference compounds it was found that the efficacy of SAC (a)
Åꢀ
Å
ꢀ
1
to scavenge O , H O , OH , and ONOO was lower, (b) to scavenge HOCl was similar, and (c) to scavenge O was higher. In addition,
2
2
2
2
it was found that SAC was able to prevent K
2
Cr
2
O
7
-induced toxicity in LLC-PK1 cells in culture. It was showed for the first time that
1
SAC is able to scavenge O
2 2 2 7
and HOCl and to ameliorate the K Cr O -induced toxicity.
ꢀ
2007 Elsevier Ltd. All rights reserved.
2 2 7
Keywords: S-allylcysteine; Scavenging activity; Peroxynitrite; Singlet oxygen; Hypochlorous acid; Oxidative stress; K Cr O
1
. Introduction
among the organosulfur compounds measured in a com-
mercial preparation of aged garlic extract (0.62 mg/g) and
after 720 days of aging of chopped garlic in 20% ethanol
(7.2 mg/g) (Lawson, 1996). It has been clearly shown that
SAC has antioxidant properties both in vivo (Numa-
gami et al., 1996; Numagami and Ohnishi, 2001; Mostafa
et al., 2000; Maldonado et al., 2003; Perez-Severiano
et al., 2004a,b; Herrera-Mundo et al., 2006; Kim et al.,
2006a,b) and in vitro (Kim et al., 2001; Yamasaki et al.,
1994; Ho et al., 2001; Ide and Lau, 1997, 1999, 2001; Geng
S-allylcysteine (SAC) (Nagae et al., 1994) is a water-
soluble and non-toxic garlic compound and the most abun-
dant organosulfur compound in aged garlic extracts (AGE)
(
Lawson, 1996). The concentration of SAC was the highest
*
Corresponding author. Tel./fax: +52 55 5622 3878.
E-mail address: pedraza@servidor.unam.mx (J. Pedraza-Chaverri).
0
278-6915/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2007.05.002

O.N. Medina-Campos et al. / Food and Chemical Toxicology 45 (2007) 2030–2039
2031
et al., 1997; Ide et al., 1997; Imai et al., 1994; Kim et al.,
006a,b). In vivo, SAC ameliorates (a) edema formation
To our knowledge, it has not been explored the potential
protective effect of SAC on K Cr O -induced nephro-
2
2
2
7
in the ischemic rat brain, by a mechanism that seems to
involve the inhibition of lipid peroxidation (Numagami
et al., 1996; Numagami and Ohnishi, 2001), (b) hippocam-
pal damage induced by ischemia and reperfusion in gerbils
toxicity.
Interestingly, the potential ability of SAC to scavenge
singlet oxygen ( O ) and hypochlorous acid (HOCl) has
not been studied. In addition the IC50 values of SAC for
each one of the reactive oxygen species (ROS) have not
been calculated. Based on the above mentioned informa-
1
2
(
Kim et al., 2006a), (c) the histological damage in heart and
liver of mice treated with doxorubicin, an anticancer drug
Mostafa et al., 2000), (d) gentamicin-induced nephrotoxi-
(
tion, the purposes of this work was (a) to study if SAC
has the ability to scavenge O and HOCl, (b) to further
1
city and oxidative stress (Maldonado et al., 2003), (e) oxi-
dative damage and learning deficits induced by amyloid-
beta peptide (Perez-Severiano et al., 2004b), (f) quinolinic
acid-induced neurotoxicity (Perez-Severiano et al., 2004a),
2
characterize the scavenging properties of SAC against
Åꢀ
Å
ꢀ
O , H O , OH , and ONOO , and (c) to know if SAC
2
2
2
is able to prevent K Cr O -induced toxicity in LLC-PK1
2
2
7
(
g) 3-nitropropionic acid-induced neurotoxicity and oxida-
cells. The ROS scavenging properties were compared
against those of reference compounds.
tive stress (Herrera-Mundo et al., 2006), (h) acetamino-
phen-induced hepatotoxicity in mice (Mizuguchi et al.,
2
006), (i) carbon tetrachloride-induced oxidative stress
2
. Materials and methods
and pulmonary fibrosis in rats (Hsu et al., 2006), and (j)
learning deficits in senescence-accelerated mice (Nishiyama
et al., 2001). Furthermore, Hsu et al. (2004) have found
that SAC has antioxidant activity in Balb/cA mice which
2
.1. Reagents
N-acetylcysteine (NAC), L-cysteine, glutathione (GSH), sodium pyru-
0
vate, 5,5 -dithio-bis(2-nitrobenzoic acid) (DTNB), sodium borohydride,
dimethyl thiourea (DMTU), DL-penicillamine, dimethyl sulfoxide
was measured by several parameters.
Åꢀ
In vitro, SAC is able to scavenge superoxide anion (O )
2
(
DMSO), lipoic acid, histidine, ascorbic acid, xylenol orange, butylated
(
Kim et al., 2001; Maldonado et al., 2003), H O (Ide and
2 2
hydroxytoluene, N,N-dimethyl-4-nitrosoaniline, xanthine, xanthine oxi-
dase, nitroblue tetrazolium (NBT), potassium nitrite (KNO ), manganese
dioxide (MnO
carbonate (Na CO ), allyl bromide, ammonium iron (II) sulfate hexahy-
4 2 4 2 2
drate ((NH ) Fe(SO ) 6H O), bovine serum albumin (BSA), Tris–HCl,
mercaptoethanol, bromophenol blue, Coomassie brilliant blue, were from
Sigma-Aldrich (St. Louis, MO, USA). Dihydrorhodamine 123 (DHR 123)
was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA).
Å
Lau, 2001; Maldonado et al., 2003), OH (Kim et al., 2001;
Chung, 2006), and ONOO (Kim et al., 2006b). SAC is
also able to ameliorate (a) ONOO induced hemolysis
2
ꢀ
2
), diethylene-triamine-pentaacetic acid (DTPA), sodium
ꢀ
2
3
(
Morihara et al., 2005), (b) nitropropionic acid-induced
lipid peroxidation and mitochondrial dysfunction in rat
brain synaptosomes (Perez-de la Cruz et al., 2006), (c)
H O -induced endothelial cell injury and lipid peroxidation
2 2
H O , trichloroacetic acid, ethylenediaminetetraacetic acid (EDTA), and
2
2
sodium hypochlorite (NaOCl) were from JT Baker (Xalostoc, Edo.
M e´ xico, M e´ xico). HPLC-grade methanol, and all other chemicals were
reagent grade and commercially available.
(
Yamasaki et al., 1994; Ide and Lau, 2001), (d) low-density
lipoprotein oxidation (Higuchi et al., 2003; Ide and Lau,
1
2
induced by oxygen and glucose deprivation in human neu-
roblastoma cell line (SK-N-SH) (Kim et al., 2006a). In
addition, it has been shown that SAC have protective effect
on (a) amyloid-beta peptide-induced apoptosis in vitro
997, 2001; Ho et al., 2001; Ide et al., 1997; Huang et al.,
004; Nishimura et al., 2004, 2006), and (e) the damage
2
.2. Synthesis of SAC
SAC was synthesized by the reaction of L-cysteine with allyl bromide
and purified by recrystallization from ethanol-water as previously descri-
bed (Lawson et al., 1991; Maldonado et al., 2003).
(
Peng et al., 2002), (b) neurotoxicity induced by amyloid
beta-peptide and ibotenic acid in organotypic hippocampal
cultures (Ito et al., 2003a), (c) amyloid beta-protein-induced
cell death in nerve growth factor-differentiated PC12 cells
2
.3. Superoxide anion scavenging assay
Åꢀ
Xanthine–xanthine oxidase system was used to determine the O2
Åꢀ
scavenging capacity of SAC. O2 in the assay system and xanthine oxidase
activity were measured as NBT reduction and uric acid production,
respectively (Pedraza-Chaverrı et al., 2006; Floriano-S a´ nchez et al., 2006),
using a DU-64O series Beckman spectrophotometer. This system is useful
to test for O2 scavenging capacity only when the extracts or compounds
(
Ito et al., 2003b), and (d) cultured rat hippocampal neu-
rons from amyloid beta-protein- and tunicamycin-induced
neuronal death (Kosuge et al., 2003). Furthermore, Geng
et al. (1997) have shown that SAC may block H O -
´
Åꢀ
2
2
used do not interfere with the xanthine oxidase activity. A compound with
induced nuclear factor kappa B activation.
Åꢀ
O
scavenging capacity should decrease NBT reduction without inter-
2
On the other hand, it is known that potassium dichro-
mate (K Cr O )-induced nephrotoxicity (Pedraza-Chaverri
fering with xanthine oxidase activity measured as uric acid production.
Eight hundred lL of the following reaction mixture: 0.116 mM xanthine,
2
2
7
et al., 1995) is associated with oxidative stress (Barrera
2
2 3
0 mM Na CO , 29 lM NBT, and 18 mM phosphate buffer (pH 7.0) were
´
et al., 2003a,b; Pedraza-Chaverrı et al., 2005) which has
mixed with 100 lL of 50 mM phosphate buffer (pH 7.0) (0% scavenging
tube) or with 100 lL of different concentrations of SAC (from 0 to
been supported by the fact that some antioxidants (Hojo
and Satomi, 1991; Standeven and Wetterhahn, 1991; Na
et al., 1992; Sugiyama, 1992; Appenroth and Winnefeld,
5
1
0 mM) or NAC (0–15 mM). The reaction was started by the addition of
00 lL of xanthine oxidase (168 U/L). Optical density was registered both
Åꢀ
at 295 nm (for uric acid production) and 560 nm (for O2 in the assay
system). Scavenging percent was obtained from the optical densities at
560 nm.
1
998) and the previous induction of heme oxygenase-1
are able to ameliorate K Cr O -induced nephrotoxicity.
2
2
7

2032
O.N. Medina-Campos et al. / Food and Chemical Toxicology 45 (2007) 2030–2039
2
.4. Hydrogen peroxide scavenging assay
amounts of SAC (from 0 to 6 mM) or lipoic acid (from 0 to 12 mM) or
GSH (from 0 to 13 mM).
A solution of 75 lM H
enging tube) or with different concentrations of SAC (0–125 mM) and
incubated for 30 min at room temperature. After this, H was measured
by the method described by Long et al. (1999). Briefly, 9 volumes of
.4 mM butylated hydroxytoluene in HPLC-grade methanol were mixed
with 1 volume of 1 mM xylenol orange and 2.56 mM ammonium ferrous
sulfate in 0.25 M H SO to give the ‘‘working’’ FOX reagent. Ninety ll of
the H -sample solutions were pippeted in 1.5 mL eppendorf tubes and
mixed with 10 ll of HPLC-grade methanol immediately followed by the
addition of 0.9 mL of FOX reagent, vortexed for 5 s and then incubated at
room temperature for 30 min. The tubes were centrifuged for 15,000 · g
for 10 min and the absorbance at 560 nm was read. The concentration of
2 2
O was mixed (1:1 v/v) with water (0% scav-
2
.7. Hypochlorous acid scavenging activity
2 2
O
Hypochlorous acid was prepared immediately before use by adjusting
4
the pH of a 1% (v/v) solution of NaOCl to pH 6.2 with 0.6 M sulphuric
acid. The concentration of HOCl was further determined spectrophoto-
metrically at 235 nm using the molar extinction coefficient of
2
4
2
O
2
ꢀ1 ꢀ1
1
00 M cm . 5-thio-2-nitrobenzoic acid (TNB) was prepared according
Ching et al. (1994) with some modifications. Briefly, 50 mM sodium
borohydride was added to a 1 mM solution of DTNB in 50 mM potassium
phosphate buffer, pH 6.6, containing 5 mM EDTA. The solution was
incubated at 37 ꢁC for 30 min. The concentration of TNB was determined
by measuring the absorbance at 412 nm using the molar extinction coef-
H
2
O
2
was calculated from a standard curve prepared with increasing H
concentrations. Sodium pyruvate (0–0.1 mM) was used as standard for
scavenging (Floriano-S a´ nchez et al., 2006; Pedraza-Chaverrı et al.,
2 2
O
ꢀ1
ꢀ1
ficient of 13,600 M cm . The assay for scavenging HOCl was per-
formed as follows (Fernandes et al., 2003). The 0% scavenging value was
obtained from the difference in optical density of a tube containing 0.4 ml
of 175 lM TNB, 0.1 ml of distilled water and 0.5 ml of 50 mM potassium
phosphate pH 6.6, and of a tube containing 0.4 ml of TNB 175 lM, 0.1 ml
of distilled water and 0.5 ml of HOCl 50 lM that were vortexed, incubated
at room temperature for 5 min and read at 412 nm against 50 mM
potassium phosphate buffer, pH 6.6. The scavenging activity was esti-
mated from the difference in the absorbance of tubes with SAC (0–25 mM)
or lipoic acid (0–20 mM) instead of water. The difference in the optical
density of tubes with SAC or lipoic acid was compared against the dif-
ference in the optical density of tubes with water and the data are
expressed as the percentage of inhibition of the TNB oxidation to DTNB.
2 2
H O
´
2006).
2
.5. Hydroxyl radical scavenging assay
Å
Experiments of OH -mediated oxidation of BSA were carried out by
using a metal-catalyzed reaction based on Kocha et al. (1997) with
modifications. A solution of ascorbic acid (0.8 mM)/EDTA (0.4 mM)/
(
7
NH
.4 and the BSA, SAC (0–5 mM) and DMTU (0–4 mM) were dissolved in
it. The assay was made as follows: In a 1.5 mL eppendorf tubes 250 lL of
% BSA were mixed with 250 lL of the same solution of ascorbic acid/
4 2 4 2
) Fe(SO ) (0.4 mM) was prepared in 50 mM phosphate buffer, pH
1
EDTA/(NH
4
)
2
Fe(SO
4
)
2
(with SAC or DMTU). Tubes without SAC or
Å
2.8. Peroxynitrite scavenging activity
DMTU were considered as 0% scavenging tubes. The generation of OH
was initiated with the addition of 15 lL of 2% H
2
O
2
. In the control tube
Å
Peroxynitrite was synthesized as previously described (Beckman et al.,
(without generation of OH ), the H
O
2 2
was replaced by water. After
1
994). Five mL of an acidic solution (0.6 M HCl) of H
mixed with 5 mL of 0.6 M KNO on ice bath for 1 s and the reaction
was quenched with 5 mL of ice-cold 1.2 M NaOH. Residual H was
removed using granular MnO prewashed with 1.2 M NaOH and the
2 2
O (0.7 M) was
30 min of incubation at room temperature, 250 lL of 20% trichloroacetic
2
acid was added and the mixture was centrifuged at 2236 · g/30 min/4 ꢁC.
The supernatant was discarded and the pellet was resuspended in 500 lL
of 0.1 M NaOH.
2
O
2
2
Å
reaction mixture was then left overnight at ꢀ20 ꢁC. The resulting yellow
To determinate protein damage by OH , the samples were subjected to
liquid layer on the top of the frozen mixture was collected for the exper-
SDS-PAGE. Forty micrograms of BSA (from resuspended pellet) were
mixed 1:1 with loading buffer (10% glycerol, 2% SDS, 25 mM Tris–HCl
ꢀ
iment. Concentrations of ONOO were determined before each experi-
ꢀ1
ꢀ1
ment at 302 nm using a molar extinction coefficient of 1,670 M cm .
(pH 6.8), 5% mercaptoethanol, 0.1% bromophenol blue) and heated at
Peroxynitrite scavenging activity was measured by monitoring the oxida-
tion of DHR 123 by a modified method of Kooy et al. (1994). A working
solution of 50 lM of DHR 123 diluted from the stock solution (28 mM
DHR 123 in DMSO) was placed on ice in the dark immediately before the
assay. The reaction mixture (in a final volume of 0.5 mL in 0.1 M phos-
phate buffer, pH 7.4) consisted of 10 lM DTPA, 5 lM DHR 123, samples
1
00 ꢁC for 1 min. The protein sample was loaded in a 12.5% polyacryl-
amide gel and electrophoresed at 100 V. After running, gels were stained
with 0.2% Coomassie brilliant blue for 1 h, destained and scanned in HP
scanner model 2200c (Hewlett Packard Company, Palo Alto, CA, USA)
for densitometric analysis with SigmaScan Pro software (version 4.01;
SPSS Inc. Chicago, IL, USA). The area of each band was obtained and
compared respect to control band (BSA neither SAC nor DMTU and
ꢀ
at different concentrations, and 25 lM ONOO . The optical density at
5
00 nm was recorded. The tube with the reaction mixture and without
without H
and with H
2
O
2
) and 0% scavenging band (BSA neither SAC nor DMTU
ꢀ
sample was considered as 0% of ONOO scavenging capacity or 100% of
DHR 123 oxidation by the ONOO added to assay. DL-Penicillamine (0–
2
O ).
2
ꢀ
0
.1 mM) was used as reference compound (Floriano-S a´ nchez et al., 2006).
2
.6. Singlet oxygen assay
2
.9. Determination of scavenging capacity and IC50 values
1
The production of O
2 2 2
by NaOCl and H O was determined using a
previously reported spectrophotometric method (Chakraborty and Tri-
Scavenging capacity was expressed as 50% of the inhibitory concen-
´
pathy, 1992; Floriano-S a´ nchez et al., 2006; Pedraza-Chaverrı et al., 2004)
tration (IC50) value, which denotes the concentration of SAC or reference
with minor modifications in which N,N-dimethyl-p-nitrosoaniline was
compound (mM) required to give a 50% reduction in (a) NBT reduction
1
Åꢀ
used as a selective acceptor of
O
2
. The bleaching of N,N-dimethyl-p-
(for O ), (b) H
2
O
2
concentration (for H
2
O
2
), (c) albumin degradation (for
2
Å
1
nitrosoaniline was monitored spectrophotometrically at 440 nm. The
assay mixture contained 45 mM sodium phosphate buffer (pH 7.1),
OH ), (d) the bleaching of N,N-dimethyl-p-nitrosoaniline (for
O
2
), (e)
ꢀ
TNB oxidation (for HOCl), and (f) DHR 123 oxidation (for ONOO ),
relative to the tube without SAC or reference compound. In other words,
the IC50 value is the concentration of SAC that is able to scavenge or to
inhibit 50% of the ROS present or generated in each specific assay system.
IC50 values were calculated according to this definition in spite that in
several cases the scavenging activity of SAC does not reach 100% with the
higher concentration tested in the assay. Diverse authors have calculated
IC50 values in this way (Yu et al., 2005; Lass et al., 2002; Conforti et al.,
10 mM histidine, 10 mM NaOCl, 10 mM H
2 2
O , and 50 lM N,N-dimethyl-
p-nitrosoaniline. The total reaction volume was 2.0 mL, and it was incu-
1
2
bated at 30 ꢁC for 40 min. The extent of O production was determined
by measuring the decrease in the absorbance of N,N-dimethyl-p-nitro-
soaniline at 440 nm. The relative scavenging efficiency (% inhibition in
1
production of O
2
) was estimated from the difference in the absorbance of
N,N-dimethyl-p-nitrosoaniline with and without the addition of increasing

O.N. Medina-Campos et al. / Food and Chemical Toxicology 45 (2007) 2030–2039
006; Barthomeuf et al., 2001; Puttaraju et al., 2006). In the present study Table 1
2033
2
IC50 was calculated by the least square method. The lower the IC50 value
the higher the scavenging capacity of the compound.
Scavenging activity of ROS (IC50 values) of SAC and reference
compounds
Reactive oxygen specie
Compound
IC50 (mM)
2
.10. Cell culture and treatments
Superoxide anion ðO2Åꢀ
Þ
SAC
NAC
SAC
Pyruvate
SAC
DMTU
SAC
Lipoic acid
SAC
Lipoic acid
GSH
SAC
14.49 ± 1.67(4)
3.39 ± 0.14(8)
68 ± 1.92(3)
*
*
*
**
*
LLC-PK1 cells (ATCC, Manassas, VA, USA) were maintained in
Hydrogen peroxide (H
2
O
2
)
Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO Invitrogen,
Carlsbad, CA, USA), supplemented with 10% fetal bovine serum, 100
units/mL penicillin, and 100 lg/ml streptomycin. LLC-PK1 cells grown in
2.83 ± 0.29(6)
0.68 ± 0.06(5)
0.47 ± 0.06(4)
2.86 ± 0.15(8)
2.29 ± 0.22(4)
1.93 ± 0.27(4)
4.07 ± 0.17(6)
6.99 ± 0.23(6)
Å
Hydroxyl radical (OH )
96-well plates were incubated with (a) 0, 2 or 20 mM SAC 6 h before and
Hypochlorous acid (HOCl)
along with 0 or 40 lM of K
along with 0 or 40 lM of K
2
Cr
Cr
2
O
7
for 24 h or (b) 0, 2 or 20 mM SAC
for 24 h.
2
2
O
7
1
Singlet oxygen ( O
2
)
*
2
.11. Cell viability
The crystal violet assay, based on the inability of dead cells to remain
**
ꢀ
Peroxynitrite (ONOO )
0.80 ± 0.05(10)
0.03 ± 0.002(8)
***
Penicillamine
adherent to cell culture plastic (Flick and Gifford, 1984), was used for cell
viability determination. After treatment, cells were washed with phosphate
buffered saline pH 7.4 to remove dead cells (nonadherent cells). The
remaining adherent (viable cells) were fixed with 1.1% (v/v) glutaraldehyde
and stained with 0.5% crystal violet solution at room temperature for
Data are mean ± SEM. Data in parenthesis indicate number of indepen-
dent assays.
SAC, S-allylcysteine; NAC, N-acetycysteine; GSH, reduced glutathione;
DMTU, dimethylthiourea.
*
p < 0.05.
p < 0.001.
p < 0.0001 vs. SAC.
15 min. The plates were thoroughly washed with water and air-dried. The
*
*
crystal violet was dissolved in 10% acetic acid. The absorbance of dis-
solved dye was measured in an automated microplate reader at 595 nm.
Cell viability was expressed as a percentage of the control optical density,
deemed as 100%.
*
**
efficiently than SAC. In addition, Fig. 1 shows that SAC
was unable to affect the uric acid production indicating that
the decrease in NBT reduction was secondary to the O2
2
.12. Statistics
Åꢀ
Data are expressed as mean ± SEM. The scavenging capacity was
scavenging ability and not to the inhibition of xanthine oxi-
dase activity. Fig. 2 shows the H O scavenging capacity of
compared against the tube with 0% scavenging capacity by one way
analysis of variance (ANOVA) followed by Dunnett post-test. The IC50
values were compared by non-paired t test or by ANOVA and the Bon-
ferroni t post-test, as appropriate. Data from cell culture were compared
by ANOVA followed by Bonferroni t-test. p < 0.05 was considered
significant.
2
2
SAC in a concentration-dependent way. The scavenging
capacity was significant on 12.5–125 mM of SAC (Fig. 2).
The calculated IC50 of SAC was of 68 ± 1.92 mM which
was higher than that obtained for the reference compound
sodium pyruvate (2.83 ± 0.29 mM) (Table 1) indicating
that this compound scavenges H O more efficiently than
3
. Results
Fig. 1 shows that SAC was able to scavenge O2 in a con-
2
2
SAC.
Åꢀ
Å
Figs. 3 and 4 show the OH scavenging capacity of SAC
in a concentration-dependent way. Fig. 4 shows that SAC
protected albumin from OH -induced degradation in a con-
centration-dependent way. The scavenging capacity was
significant on 3–50 mM of SAC (Fig. 1) and the calculated
IC50 was of 14.5 ± 1.67 mM which was higher than that of
Å
centration-dependent way. Lane 2 shows the extent to
Å
the reference compound NAC (IC50 = 3.39 ± 0.14 mM)
which albumin is degraded by OH in absence of SAC
Åꢀ
(
Table 1) indicating that this compound scavenges O2 more
Åꢀ
Fig. 1. O2 scavenging capacity of SAC. Data are presented as mean ±
2 2
Fig. 2. H O scavenging capacity of SAC. Data are presented as
*
*
SEM, n = 4. p < 0.05 vs. 0 mM. IC50 = 14.5 ± 1.67 mM.
mean ± SEM, n = 3. p < 0.001 vs. 0 mM. IC50 = 68 ± 1.92 mM.

2034
O.N. Medina-Campos et al. / Food and Chemical Toxicology 45 (2007) 2030–2039
Å
Fig. 5. Representative SDS-PAGE electrophoresis for OH scavenging
assay. Details of the assay are indicated in material and methods section.
Å
Lane 1: BSA, Lane 2: BSA + OH without DMTU, Lane 3:
Å
Å
BSA + OH + DMTU 8 mM, Lane 4: BSA + OH + DMTU 4 mM, Lane
Å
Å
5
: BSA + OH + DMTU 2 mM, Lane 6: BSA + OH + DMTU 1 mM,
Lane 7: BSA + OH + DMTU 0.25 mM, Lane 8: BSA + OH + DMTU
.05 mM.
Å
Å
0
Å
Fig. 3. OH scavenging capacity of SAC. Data are presented as mean ±
*
SEM, n = 5. p < 0.001 vs. 0 mM. IC50 = 0.68 ± 0.06 mM.
increases as DTMU concentration decreases. This garlic
compound was also able to scavenge HOCl in a concentra-
tion-dependent way (Fig. 6). The scavenging activity was
significant on 0.1–25 mM of SAC. The calculated IC50
was of 2.9 ± 0.149 mM which was similar to that of the ref-
erence compound lipoic acid (2.29 ± 0.22 mM) (Table 1)
indicating that this compound has a similar efficacy to
scavenge HOCl that SAC. Fig. 7 shows that SAC was able
1
to scavenge O in a concentration-dependent way. The
2
Å
Fig. 4. Representative SDS-PAGE electrophoresis for OH scavenging
assay. Details of the assay are indicated in material and methods section.
BSA and SAC were dissolved in solution of ascorbic acid (0.8 mM)/
EDTA (0.4 mM)/(NH ) Fe(SO ) (0.4 mM) that was prepared in 50 mM
4 2 4 2
phosphate buffer, pH 7.4. The electrophoresis show that the BSA without
Å
H
2
O
2
addition (without OH generation) remains intact (band 1), but in
Å
presence of H
2
O
2
(with the consequent formation of OH ) the decrease in
the intensity of the BSA band is evident indicating the OH -induced
damage. When SAC is added to the assay this damage to BSA is prevented
in a concentration-dependent way. Lane 1: BSA, Lane 2: BSA + OH
without SAC, Lane 3: BSA + OH + SAC 5 mM, Lane 4:
Å
Å
Fig. 6. HOCl scavenging capacity of SAC. Data are presented as
Å
*
mean ± SEM, n = 8. p < 0.001 vs. 0 mM. IC50 = 2.9 ± 0.149 mM.
Å
Å
BSA + OH + SAC 1.7 mM, Lane 5: BSA + OH + SAC 0.6 mM, and
Lane 6: BSA + OH + SAC 0.2 mM.
Å
and lanes 3–6 show the extent of albumin protection with
decreasing amounts of SAC. It is clear that albumin degra-
dation increases as SAC concentration decreases. The scav-
enging capacity was significant on 0.2–5 mM of SAC. The
calculated IC50 was of 0.68 ± 0.06 mM which was slightly
higher than that obtained for the reference compound
DMTU (IC50 = 0.47 ± 0.06 mM) (Table 1 and Fig. 5) indi-
Å
cating that this compound scavenges OH more efficiently
than SAC. Fig. 5 shows that DMTU protected albumin
Å
from OH -induced degradation in a concentration-depen-
dent way. Lane 2 shows the extent to which albumin is
Å
degraded by OH in absence of DMTU and lanes 3–8 show
the extent of albumin protection with decreasing amounts
of DMTU. It is clear that the albumin degradation
1
Fig. 7.
2
O scavenging capacity of SAC. Data are presented as mean ±
*
SEM, n = 4. p < 0.001 vs. 0 mM. IC50 = 1.933 ± 0.271 mM.

O.N. Medina-Campos et al. / Food and Chemical Toxicology 45 (2007) 2030–2039
2035
scavenging activity was significant on 1–6 mM of SAC. The
calculated IC50 was of 1.933 ± 0.271 mM which was lower
than the reference compounds lipoic acid and GSH which
presented values of IC50 of 4.07 ± 0.17 and 6.99 ±
bility to 107.8 ± 1.73% and 111.3 ± 0.94%, respectively.
When SAC (2 mM or 20 mM) was incubated along
K Cr O , cell viability increased significantly (p < 0.001)
2
2
7
to 77.88 ± 1.58% and 82.60 ± 2.85%.
0
.23 mM, respectively (Table 1). These data indicate that
1
the ability of SAC to scavenge O was higher than that
4. Discussion
2
of lipoic acid and GSH. SAC has also the ability to scav-
enge in a concentration-dependent way the reactive nitro-
gen specie ONOO (Fig. 8). This scavenging ability was
Our data clearly indicates for the first time that SAC is
ꢀ
1
able to scavenge in a concentration-dependent way O and
2
significant on 0.4–2.0 mM of SAC. The calculated IC50
was of 0.8 ± 0.05 mM which was higher than that obtained
for penicillamine (0.03 ± 0.002 mM) indicating that this
HOCl and to protect LLC-PK1 cells from K Cr O -
induced toxicity which has been associated to oxidative
stress. It is remarkable that SAC was as efficient to scav-
2
2
7
ꢀ
compound scavenges ONOO more efficiently than SAC.
enge HOCl as the reference compound lipoic acid and more
1
Fig. 9 shows that SAC was able to ameliorate K Cr O -
efficient to scavenge O than the reference compounds
2
2
7
2
induced by cell damage in LLC-PK1 cells either at 2 or
0 mM compared to control cells which were incubated
in absence of SAC and K Cr O . K Cr O decreased cell
lipoic acid and GSH. Interestingly, it has been previously
2
found that the structural related garlic compound, S-allyl-
1
mercaptocysteine, is also able to scavenge O in a concen-
2
2
7
2
2
7
2
viability from 100 ± 0.73% in control cells to 28.8 ±
tration-dependent way with an IC5 value close to 1 mM
0
1
1
2
.79%. Incubation with 2 or 20 mM of SAC alone by
4 h increased slightly but significantly (p < 0.05) cell via-
(Pedraza-Chaverr ´ı et al., 2004). O , an electronically
2
excited species of oxygen, has been reported to be gener-
ated in myeloperoxidase- and eosinophil peroxidase-cata-
lyzed reactions and by certain activated cells including
neutrophils, eosinophils and macrophages. Direct exposure
of a number of compounds to ultraviolet light has been
1
shown to generate O . As a result of the exposure of
2
1
humans to UV and visible light, O has been suggested
2
to play a key role in the development of a number of
human pathologies including aging, some skin cancers,
1
sunburn and cataract. O can adversely alter several bio-
2
logical molecules including lipids, DNA, amino acids,
and proteins (Morgan et al., 2004). HOCl, or its anion
ꢀ
hypochlorite ( OCl), is produced in vivo by activated
phagocytes in myeloperoxidase–hydrogen peroxide-chlo-
ride ion system and it is responsible for the bacterial cell
killing action of neutrophils and macrophages. Hypochlo-
rite is a strong oxidant and reacts quickly with thiols and
amines, thus modifying proteins and enzymes (Noguchi
ꢀ
Fig. 8. ONOO scavenging capacity of SAC. Data are presented as
*
mean ± SEM, n = 10. p < 0.001 vs. 0 mM. IC50 = 0.68 ± 0.06 mM.
et al., 2002). Our data suggest that SAC may ameliorate
1
diseases associated with excessive production of O and
2
HOCl.
Åꢀ
Å
In addition our data confirm the O , H O , OH , and
2
2
2
ꢀ
ONOO scavenging ability of this garlic compound
although we additionally found that this effect was concen-
tration-dependent which allowed us to calculate IC50
values for the first time. The ability of SAC to scavenge
Åꢀ
ꢀ
O , H O , and ONOO was clearly lower than those
2
2
2
observed for the reference compounds NAC, sodium pyru-
vate and penicillamine, respectively. In contrast, the ability
Å
of SAC to scavenge OH was lower but very close to that of
the reference compound DMTU.
Åꢀ
Living organisms produce large quantities of O , a
2
relatively benign radical. Although, it readily inactivates
iron–sulfur proteins, it does not react with most biological
molecules. However, the iron released from iron–sulfur
Fig. 9. Effect of SAC on K
Cellular viability was measured by the crystal violet method 24 h after the
Cr (40 lM) addition. Control cells were incubated without SAC and
without K Cr . Cells were coincubated with SAC and K Cr by 24 h.
Data are mean ± SEM, n = 10. *p < 0.05 vs. control, #p < 0.001 vs.
Cr
2 2 7
Cr O -induced cell damage in LLC-PK1 cells.
K
2
2 7
O
Å
proteins can give rise to secondary products such as OH
2
O
2 7
2
2 7
O
ꢀ
which together with ONOO are thought to be major con-
Åꢀ
Åꢀ
Å
K
2
O
2 7
.
tributors of O2 toxicity. O2 reacts with nitric oxide (NO )

2036
O.N. Medina-Campos et al. / Food and Chemical Toxicology 45 (2007) 2030–2039
ꢀ
to generate ONOO . This reaction is extremely quick and
1994; Ide and Lau, 2001) and with the blocking effect of
SAC on H O -induced nuclear factor kappa B activation
ꢀ
ONOO is capable of undergoing a wide range of oxidative
2
2
processes. These include direct oxidations and secondary
reactions due to nitrogen dioxide, hydroxyl and carbonate
radicals (reviewed by Winterbourn and Kettle, 2003). Fur-
(Geng et al., 1997) and DNA damage in HepG2 cells (Bell-
oir et al., 2006).
The cytoprotective effect of SAC has been observed not
only in the mM order (Peng et al., 2002; Ide and Lau, 2001)
but also in the lM order (Kim et al., 2001, 2006a,b; Ito
et al., 2003a,b). Peng et al. (2002) found that SAC (3.1–
12.8 mM and preincubated for 24 h) was able to prevent
beta-amyloid peptide induced apoptosis, cell death and
ROS production in PC12 cells. Ide and Lau (2001)
observed that SAC (0.1–20 mM) was able to protect pul-
monary artery bovine endothelial cells from oxidized-low
density lipoprotein-induced cell death, lipid peroxidation
and decrease in intracellular GSH. In addition Kim et al.
(2001) found that SAC (up to 80 lM) was able to prevent
the lipopolysaccharide-interferon c-induced inducible nitric
oxide synthase mRNA expression and nitrite production in
murine macrophage RAW 264.7 cells and Kim et al.
(2006a) found that preincubation by 48 h with SAC (10–
100 lM) prevented cell death induced by oxygen-glucose
deprivation in human neuroblastoma cells (SK-N-SH).
It has been found that SAC (10 or 100 lM) was able to
prevent cell death induced by beta amyloid peptide and
ibotenic acid in hippocampal neurons (Ito et al., 2003a)
and PC12 cells (Ito et al., 2003b). Ito et al. (2003b) also
observed that SAC (0.001–10 lM) was able to prevent
tunicamycin-induced cell death in PC12 cells.
ꢀ
thermore, ONOO may oxidize and covalently modify all
major types of host biomolecules, including DNA, protein,
ꢀ
and lipids. ONOO -induced modification of proteins has
been connected with the development of several diseases
Å
(
Szabo, 2003; Denicola and Radi, 2005). OH , which is
formed via the Fenton reaction in living systems, is the
Å
most reactive oxygen radical. The half-life of OH in cells
Å
has been estimated to be around 10 s. OH is generally con-
sidered to be a harmful byproduct of oxidative metabolism,
causing molecular damage in living systems. These radicals
have been postulated to play a role in several pathological
conditions, such as carcinogenesis, cardiovascular disease,
Parkinson’s disease, rheumatoid arthritis, brain ischemia,
etc. (reviewed by Cheng et al., 2002).
Interestingly, evidences suggest that ROS are involved in
Cr(VI)-induced cell injury (Sengupta et al., 1992; Liu and
Shi, 2001; Stohs and Bagchi, 1995; Bagchi et al., 2002).
Chromium reduction intermediates Cr(V), Cr(IV), and
Cr(III), may be toxic as they involve ROS production
(
Stohs et al., 2000; Shi and Dalal, 1990, 1994; O’Brien
and Kortenkamp, 1994) which may be generated during
physiological conditions. In vitro chromate reduction via
Å
H O has been shown to produce OH via a Fenton-like
2
2
reaction (O’Brien and Kortenkamp, 1994; Aiyar et al.,
Kim et al. (2006b) found that SAC (10 lM) significantly
reduced nitrotyrosine immunoreactivity in immunostimu-
lated/glucose-deprived astrocytes. The protective effect
against H O -induced cytotoxicity was observed with
1
1
991; Shi and Dalal, 1990; Liu et al., 1997; Tsou et al.,
996). The OH scavenging of SAC may contribute to the
Å
protective effect of this garlic compound on K Cr O
2
2
7
2
2
induced cell damage in LL-CPK1 cells. In addition, we
have previously found that nitrotyrosine staining, a foot-
print of excessive production of reactive nitrogen species
100 lM of SAC (Kim et al., 2006b). Interestingly, cytopro-
tection is not observed at lM order with all the insults.
Kim et al. (2006b) observed that SAC (up to 1000 lM)
was unable to prevent NMDA toxicity in cerebrocortical
neurons and Ippoushi et al. (2002) found that SAC
(25 lM) was unable to prevent the nitric oxide production
induced by LPS in macrophages. The potential protective
effects may depend not only on the antioxidant properties
of the compound but also of the type and the concentration
of the insult, the time of exposure to the insult and/or to
the antioxidant, and the type of cell studied.
In our study in LLC-PK1 cells it was observed lack of
cytotoxicity and cytoprotection of SAC in the mM order.
This is consistent with Kodera et al. (2002) who found that
SAC toxicity is very low. Additional desirable characteris-
tics of SAC as a good antioxidant are its high biodispon-
ibility and stability (Kodera et al., 2002).
ꢀ
including ONOO , is increased in K Cr O -induced neph-
2
2
7
rotoxicity suggesting the participation of reactive nitrogen
ꢀ
species in the K Cr O -induced cell toxicity. The ONOO
2
2
7
scavenging ability of SAC may contribute to explain the
protective effect of SAC in LLC-PK1 cells treated with
K Cr O in this study as well as the protective effect of
2
2
7
SAC observed in another experimental models associated
with enhanced nitrotyrosine such as the renal damage
induced by gentamicin (Maldonado et al., 2003). This abil-
ꢀ
ity also may contribute to the ONOO scavenging ability
of several garlic preparations observed recently (Pedraza-
´
Chaverrı et al., 2007). It is interesting to note that the
extent of SAC-induced protection of K Cr O -induced
2
2
7
toxicity in LLC-PK1 is similar with 2 or 20 mM suggesting
that SAC protection is complete with 2 mM. Taking into
In summary, we have reported for the first time that
Å
ꢀ
1
account that the IC50 values of SAC for OH and ONOO
SAC is able to scavenge O and HOCl and to prevent
2
are lower than 2 mM (0.68 ± 0.05 and 0.8 ± 0.05 mM,
K Cr O -induced toxicity in LLC-PK1 cells. In addition,
2
2
7
ꢀ
Åꢀ
Å
respectively), the above data may suggest that ONOO
the ability of SAC to scavenge O , H O , OH , and
2
2 2
Å
ꢀ
1
Åꢀ
2
and/or OH are mainly involved in this toxicity.
ONOO was also characterized. The O , HOCl, O ,
2
Å
ꢀ
The H O scavenging activity of SAC is consistent with
H O , OH , and ONOO scavenging properties of SAC
2 2
2
2
the ameliorative effect of SAC on H O -induced endothe-
lial cell injury and lipid peroxidation (Yamasaki et al.,
may contribute to explain the antioxidant properties of this
garlic compound observed in several experimental models
2
2

O.N. Medina-Campos et al. / Food and Chemical Toxicology 45 (2007) 2030–2039
2037
Floriano-Sa
´
nchez, E., Villanueva, C., Medina-Campos, O.N., Rocha, D.,
in vivo and in vitro including the K Cr O -induced toxicity
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peroxynitrite, singlet oxygen, hydroxyl radical, superoxide anion, and
hypochlorous acid and prevents in vivo tyrosine nitration in lung. Free
Radic. Res. 40, 523–533.
observed in LLC-PK1 cells.
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