S-allylmercaptocysteine scavenges hydroxyl radical and singlet oxygen in vitro and attenuates gentamicin-induced oxidative and nitrosative stress and renal damage in vivo
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Oxidative and nitrosative stress have been involved in gentamicin-induced nephrotoxicity. The purpose of this work was to study the effect of S-allylmercaptocysteine, a garlic derived compound, on gentamicin-induced oxidative and nitrosative stress and nephrotoxicity. In addition, the in vitro reactive oxygen species scavenging properties of S-allylmercaptocysteine were studied.
S-allylmercaptocysteine was able to scavenge hydroxyl radicals and singlet oxygen in vitro. In rats treated with gentamicin (70 mg/Kg body weight, subcutaneously, every 12 h, for 4 days), renal oxidative stress was made evident by the increase in protein carbonyl content and 4-hydroxy-2-nonenal, and the nitrosative stress was made evident by the increase in 3-nitrotyrosine. In addition, gentamicin-induced nephrotoxicity was evident by the: (1) decrease in creatinine clearance and in activity of circulating glutathione peroxidase, and (2) increase in urinary excretion of N-acetyl-β-D-glucosaminidase, and (3) necrosis of proximal tubular cells. Gentamicin-induced oxidative and nitrosative stress and nephrotoxicity were attenuated by S-allylmercaptocysteine treatment (100 mg/Kg body weight, intragastrically, 24 h before the first dose of gentamicin and 50 mg/Kg body weight, intragastrically, every 12 h, for 4 days along gentamicin-treatment).
In conclusion, S-allylmercaptocysteine is able to scavenge hydroxyl radicals and singlet oxygen in vitro and to ameliorate the gentamicin-induced nephrotoxicity and oxidative and nitrosative stress in vivo.
List of abbreviations
Aged garlic extract
Blood urea nitrogen
Zn-SOD Copper,zinc-dependent superoxide dismutase
- DNPH 2
Manganese-dependent superoxide dismutase
β-nicotinamide adenine dinucleotide phosphate, reduced form
Reactive oxygen species
Reactive nitrogen species
Standard error of mean
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in several renal diseases [1, 2, 3, 4, 5, 6, 7, 8] including the renal damage induced by the antibiotic gentamicin (GM) [9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19]. GM induces superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (HO) production from renal mitochondria [13, 14]. In addition, H2O2 generation , lipoperoxidation [16, 17], and the content of nitrotyrosine [10, 11, 12], and protein carbonyl [10, 18] are increased and that of reduced glutathione is diminished  in renal cortex from GM-treated rats. Moreover, the administration of several compounds with antioxidant properties, ROS scavengers, and/or antioxidant enzymes are able to ameliorate the severity of GM-induced renal damage [9, 10, 16, 17, 18, 19]. In addition, the kidneys from GM-treated rats are more vulnerable to ROS because of they are deficient in the antioxidant enzymes Mn-superoxide dismutase (Mn-SOD) [10, 17], glutathione peroxidase (GPx) , glutathione reductase (GR) , and catalase (CAT) .
On the other hand, S-allylmercaptocysteine (SAMC) (CH2=CH-CH2-S-S-CH2-CH-NH2-COOH) is one of the water soluble organosulfur compounds found in aged garlic extract (AGE) which is obtained by ethanol extraction of sliced garlic bulbs . It has been postulated that SAMC may be one of the active compounds responsible for the protective effect of AGE observed in several experimental models associated to oxidative stress [10, 21, 22, 23, 24, 25, 26, 27]. It has been clearly shown that SAMC has in vitro [21, 22] and in vivo  antioxidant properties. SAMC is able to (a) inhibit lipoperoxidation [22, 23] and low density lipoprotein oxidation  and (b) scavenge 1,1-diphenyl-2-picrylhydrazyl radical . Moreover, SAMC pretreatment protects liver against the damage induced by acetaminophen [23, 24, 25], carbon tetrachloride [25, 26], and D-galactosamine .
Based on the above mentioned data, the hypothesis was made that SAMC could ameliorate GM-induced oxidative and nitrosative stress and renal damage. In this paper the ROS scavenging properties of SAMC, and the effect of this compound on GM-induced (a) renal damage, (b) oxidative and nitrosative stress, and (c) on activity of antioxidant enzymes (Mn-SOD, GPx, GR, and CAT) in rats are presented. Renal damage was evaluated by measuring glomerular and tubular function and by histological analysis [3, 6, 7, 10]. Nitrosative stress was evaluated by measuring nitrated proteins by immunohistochemistry using antibodies against 3-nitrotyrosine (3-NT) [1, 2, 7, 10, 11, 12]. Oxidative stress was evaluated by measuring protein carbonyl content (by immunohistochemical  and spectrophotometric methods [10, 28]) and 4-hydroxy-2-nonenal (4-HNE) protein adducts (by immunohistochemistry [29, 30]). Protein oxidation by immunohistochemistry was performed using antibodies against dinitrophenol (DNP) .
In vitro ROS scavenging properties of SAMC
In vivo studies
Body weight, food intake, and urinary volume
Body weight, food intake, and urinary volume in the 4 groups of rats studied on day 5
Body weight (g)
259 ± 2.1
252 ± 4.3
263 ± 4.7
255 ± 4.3
Food intake (g/24 h)
21.2 ± 2.3
18.0 ± 1.8
21.8 ± 0.4
18.6 ± 2.2
Urinary volume(ml/24 h)
6.5 ± 1.1
18.6 ± 2.6a
7.1 ± 1.0
22.1 ± 2.4a
Markers of glomerular and tubular damage
3-nitrotyrosine (3-NT), dinitrophenol (DNP), and 4-hydroxy-2-nonenal (4-HNE) immunostaining
Spectrophotometric assay of protein carbonyl content
Antioxidant enzymes in renal cortex
Antioxidant enzymes activity in renal cortex from the four groups of rats studied on day 5
Mn-SOD (U/mg protein)
7.4 ± 0.4
4.6 ± 0.7
7.8 ± 1.2
7.9 ± 1.4
Cu,Zn-SOD (U/mg protein)
26.7 ± 2.8
28.1 ± 2.2
24.1 ± 1.1
28.6 ± 3.4
GPx (U/mg protein)
0.16 ± 0.007
0.09 ± 0.007a
0.14 ± 0.009
0.12 ± 0.007a
GR (U/mg protein)
0.037 ± 0.002
0.03 ± 0.002a
0.039 ± 0.002
0.041 ± 0.0007b
CAT (k/mg protein)
0.55 ± 0.04
0.29 ± 0.03a
0.35 ± 0.01a
0.26 ± 0.03a
GM is an aminoglycoside widely used in clinical practice for the treatment of gram-negative infections. GM is rapidly excreted, predominantly by glomerular filtration and the reabsorption of a small but notable amount of drug by the proximal tubule results in accumulation within the renal cortex; this preferential binding is responsible for nephrotoxicity . The mechanism by which GM induces nephrotoxicity remains unknown; however, it has been postulated that oxidative and nitrosative stress are involved in this process . It has been shown that O2- , OH  and H2O2 , are involved in renal damage induced by GM. In addition, GM induces H2O2 generation by mitochondria .
Interestingly, iron chelators such as deferoxamine and 2,3 dihydrobenzoic acid are able to prevent GM-induced nephrotoxicity suggesting the toxic role of iron in this disease probably by catalyzing the OH production via the Haber-Weiss reaction . GM also induces O2- production from mesangial cells in culture . This increase was suppressed by an inhibitor of NADPH oxidase or an inhibitor of nitric oxide (NO) synthase suggesting the role of both proteins in O2- production by GM in mesangial cells . The role of OH has been evident by using OH scavengers such as dimethylthiourea, sodium benzoate and dimethyl sulfoxide . The role of O2- has been shown using exogenous administration of superoxide dismutase  or M40403 , a synthetic low molecular weight compound with SOD mimetic activity, which selectively removes O2-. In addition, it has been found that GM enhances NO generation by glomeruli and mesangial cells [35, 36].
The inhibition of NO production exacerbates GM nephrotoxicity suggesting that the enhancement of NO production is very important to prevent exacerbation of renal damage in GM-induced nephrotoxicity [37, 38]. On the other hand, NO is able to react with O2- to produce peroxynitrite anion which itself is very toxic [39, 40]. In addition, there is direct evidence that peroxynitrite is able to produce 1O2 after reacting with linoleic acid hydroperoxide , suggesting a potential 1O2-dependent mechanism that may contribute to cytotoxicity mediated by lipid hydroperoxides and peroxynitrite reactions in biological systems.
In addition, it has been appreciated that OH-like activities are generated from peroxynitrite . The production of OH is frequently proposed to occur by the metal-catalyzed Haber-Weiss reaction. We are tempting to speculate that OH in GM-induced nephrotoxicity may come from both peroxynitrite and Haber-Weiss reaction. Interestingly, the OH scavenger dimethylthiourea also attenuates GM-induced in vivo lipid peroxidation  indicating the role of OH in lipid peroxidation.
The increase in O2- and NO production in GM-induced nephrotoxicity may lead to peroxynitrite formation and could explain the increase in nitrosative stress in these animals.
We found that SAMC has in vitro scavenging properties; particularly we found that SAMC scavenges OH and 1O2 (Fig. 1). These properties could be involved in the ability of SAMC to decrease lipid peroxidation which we evaluated by measuring 4-HNE. This is an α,β unsaturated aldehyde commonly used as a marker of lipid peroxidation due to it is produced in the peroxidative metabolism of arachidonic or linoleic acids . HNE rapidly modifies proteins on several amino acids residues leading to the loss of protein functions. Antibodies against 4-HNE have been successfully used for the immunodetection of lipid peroxidation in kidney sections [2, 29]. Interestingly, SAMC was also able to decrease 3-NT immunostaing in vivo suggesting that SAMC itself (or some of the metabolites) is able to scavenge in vivo peroxynitrite and/or another RNS involved in protein nitration. Immunohistochemical detection of 3-NT is a useful marker of nitrosative stress [2, 7], since it has been shown that this compound is formed by the reaction of RNS with proteins [reviewed in ]. Surprisingly, SAMC was unable to scavenge O2- and H2O2. SAMC was also able to prevent the increase in protein oxidation (measured by the increase in protein carbonyl content). Our data suggest that SAMC could be one of the compounds of AGE that may contribute to the ability of this extract to ameliorate GM-induced nephrotoxicity . In a previous paper we found that S-allylcysteine, another compound found in AGE and structurally related to SAMC, also is able to prevent GM-induced nephrotoxicity .
The in vivo antioxidant ability of SAMC is associated with the improvement in GM-induced glomerular and tubular alterations. GM induces necrosis of proximal tubular cells but the structure of glomeruli is not altered. Vasoconstriction induced by ROS is involved in the decrease in glomerular filtration rate and ROS and RNS produce cellular injury and necrosis via several mechanisms, including lipoperoxidation and protein modification. The above information may explain why antioxidants, and SAMC in particular, are able to prevent GM-induced glomerular and tubular dysfunction. In our rats SAMC was able to ameliorate the decrease in creatinine clearance and blood GPx as well as the enhancement in BUN and in the urinary excretion of both total protein and NAG. SAMC also was able to ameliorate the histological damage. All the above data clearly indicate the ability of SAMC to ameliorate GM-induced renal damage. Interestingly, SAMC was unable to prevent the increase in urinary volume induced by GM, this could be explained, in all probability, by the fact that the histological damage still is present in GM+SAMC rats (38% of damaged tubular area). This indicates that the amelioration in the histological damage is not enough to attenuate the increase in urinary volume.
The ameliorative effect of SAMC was not due to the enhancement of endogenous antioxidant defenses. It was found that SAMC itself was unable to modify the activity of Mn-SOD, Cu,Zn-SOD, GPx and GR but it decreased CAT activity (Table 2). SAMC was able to prevent the decrease in the antioxidant enzymes Mn-SOD, GPx and GR which also could contribute to the protective effect of SAMC.
The protective effect of SAMC on GM-induced nephrotoxicity was associated with the decrease in oxidative and nitrosative stress in vivo and the preservation of Mn-SOD, GPx, and GR activities in renal cortex. Our data support that SAMC is one of the compounds of AGE with antioxidant properties in vitro and in vivo. The ability of SAMC to scavenge OH and 1O2 may explain, at least in part, the in vivo antioxidant properties of SAMC. The potential protective effect of SAMC in humans receiving GM merits study.
S-allylmercaptocysteine (SAMC, Lot # 020328) was kindly provided by Wakunaga Pharmaceutical Co., Ltd. (Hiroshima, Japan). GM was from Schering-Plough (Mexico City, Mexico). Rabbit anti-3-nitro-L-tyrosine (3-NT) polyclonal antibodies (Catalogue #06-284) were from Upstate (Lake Placid, NY, USA). Mouse anti-4-hydroxy-2-nonenal (4-HNE) monoclonal antibodies (# Catalogue 24325) were from Oxis International Inc. (Portland, OR, USA). Goat anti-dinitrophenol (DNP) polyclonal antibodies (Catalogue # J06) were from Biomeda Corporation (Foster City, CA, USA). Anti-rabbit Ig horseradish peroxidase antibody (Catalogue # NA-934) and anti-mouse Ig horseradish peroxidase antibody (Catalogue # NIF-825) were purchased from Amersham Life Sciences (Buckinghamshire, England). Donkey anti-goat horseradish peroxidase antibody (Catalogue # SC2020) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Commercial kits to measure creatinine and urea were from Spinreact (Girona, Spain). Glutathione reductase, β-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), 2,4-dinitrophenylhydrazine (DNPH), histidine, ascorbic acid, D-ribose, tetramethoxy-propane, deferoxamine mesylate, thiobarbituric acid, xylenol orange, butylated hydroxytoluene, iron (III) chloride (FeCl3), N,N-dimethyl-4-nitrosoaniline were from Sigma-Aldrich (St. Louis MO, USA). Ferrous ammonium sulfate and sodium hypochlorite were from JT Baker (Xalostoc, Edo. México, México). All other chemicals were reagent grade.
In vitro experiments
Hydroxyl radical assay
The ability of SAMC to scavenge OH was conducted in the Fe3+-EDTA-H2O2-deoxyribose system . The system contained different amounts of SAMC, or an equivalent volume of distilled water for the control, 0.2 mM ascorbic acid, 0.2 mM FeCl3, 0.208 mM EDTA, 1 mM H2O2, 0.56 mM deoxyribose, and 20 mM phosphate buffer (pH 7.4). Hydroxyl radicals were generated by incubating the mixture at 37°C for 60 min. The iron salt (FeCl3) was mixed with EDTA before addition to the reaction mixture. The extent of deoxyribose degradation by the formed OH was measured directly in the aqueous phase by the thiobarbituric acid test .
Singlet oxygen assay
The production of 1O2 by sodium hypochlorite (NaOCl) and H2O2 was determined by using a spectrophotometric method  with minor modifications in which N,N-dimethyl-p-nitrosoaniline was used as selective acceptor of 1O2. The bleaching of N,N-dimethyl-p-nitrosoaniline was monitored spectrophotometrically at 440 nm. The assay mixture contained 45 mM sodium-phosphate buffer (pH 7.1), 10 mM histidine, 10 mM NaOCl, 10 mM H2O2 and 50 μM N,N-dimethyl-p-nitrosoaniline. The total volume reaction was 2.0 ml and incubated at 30°C for 40 min. The extent of 1O2 production was determined by measuring the decrease in the absorbance of N,N-dimethyl-p-nitrosoaniline at 440 nm. The relative scavenging efficiency (% inhibition in production of 1O2) was estimated from the difference in absorbance of N,N-dimethyl-p-nitrosoaniline with and without the addition of increasing amounts of SAMC.
Superoxide anion assay
Xanthine-xanthine oxidase system was used to determine the O2- scavenging activity of SAMC. O2- production and xanthine oxidase activity were measured as NBT reduction and uric acid production, respectively  using a DU-64O series Beckman spectrophotometer. Tubes without SAMC were taken as 100% of NBT reduction.
Hydrogen peroxide assay
A solution of 75 μM H2O2 was mixed with different concentrations of SAMC (1:1 v/v) and incubated for 3.5 h at room temperature. After this, H2O2 was measured by the method described by Long et al. . Briefly, 9 volumes of 4.4 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 H2SO4 to give the "working" FOX reagent. Ninety microliters of the H2O2-SAMC solutions were pippeted in 1.5 mL Eppendorf tubes and mixed with 0.01 mL of HPLC-grade methanol immediately followed by the addition of 0.9 mL of FOX reagent, vortexed for 5 seconds and then incubated at room temperature for 10 minutes. The tubes were centrifuged for 15,000 × g for 10 minutes and absorbance at 560 nm was reading against methanol blank.
In vivo experiments
Male Wistar rats (Harlan Teklad, México City, México) initially weighing 240–250 g were used. Experimental work was approved by CONACYT (#40009-M) and followed the guidelines of Norma Oficial Mexicana (NOM-ECOL-087-1995). All animals had free access to water and commercial rodent diet (Harlan Teklad, catalogue 2018S), and were randomly divided in four groups (n = 5 rats/group) as follows: (1) CT, injected subcutaneously (s.c.) with saline and intragastrically (i.g.) with carboxymethylcellulose 0.5%, (2) GM, treated s.c. with GM and i.g. with carboxymethylcellulose 0.5%; (3) SAMC, treated with SAMC; and (4) GM+SAMC, treated with GM and with SAMC. GM treated rats received 70 mg/Kg b.w./s.c./12 h for 4 days and SAMC treated rats received a single dose of 100 mg/Kg b.w./i.g. 24 h before the first dose of GM or saline, and 50 mg/kg b.w./i.g./12 h for 4 days alongside GM-treatment or saline . SAMC was given 30 minutes before every GM injection. SAMC was suspended in 0.5% carboxymethylcellulose at a 10 mg/ml concentration. Rats received 5.0 ml/Kg/12 h (50 mg SAMC/Kg/12 h). The dose of SAMC was chosen from the previous work of Sumioka et al. . During the study rats were maintained with a 12-h light:dark cycle in stainless steel metabolic cages to collect urine. On day 5, the animals were sacrificed by decapitation and blood was collected to obtain serum and measure creatinine, BUN and GPx activity. NAG and total protein were measured in 24-h urine. One kidney was removed to obtain cortex samples for histological and immunohistochemical studies and the other kidney was removed to obtain cortex and measure the activity of total SOD, Mn-SOD, CAT, GPx, and GR as well as the carbonyl content of proteins.
Markers of glomerular and tubular damage
The markers of glomerular damage, creatinine and urea, were measured using commercial kits. Creatinine clearance was calculated with the standard formula , and BUN was obtained by correcting the urea value by a 2.14 factor . As markers of tubular damage, we measured urinary excretion of NAG and total protein and blood GPx activity. NAG activity was measured using p-nitrophenyl-N-acetyl-β-D-glucosaminide as substrate , total protein was measured by a turbidimetric method , and blood GPx activity was measured using GR and NADPH in a coupled reaction .
Thin slices of kidney tissue with cortex and medulla were fixed by immersion in buffered formalin (pH 7.4), dehydrated and embedded in paraffin. Sections (4 μm) were stained with hematoxilin and eosin (H&E) . The histological profile of twenty proximal tubules randomly selected per rat (5 rats per experimental group) was recorded, using a Leica Qwin Image Analyzer (Cambridge, England). The percentage of tubular area with histopathological alterations like swelling, cytoplasmic vacuolization, desquamation or necrosis was obtained. The percentage of damaged area of GM and GM+SAMC groups was compared.
Protein carbonyl content
Protein carbonyl groups are relatively stable marker of protein oxidation by ROS . Carbonyl moieties in kidney tissues from all experimental groups were assessed using both spectrophotometric and immunohistochemical detection of the formation of the stable dinitrophenyl (DNP) hydrazone product which is formed when protein carbonyl groups reacts with DNPH. The spectrophotometric method has been described previously  and the immunohistochemical method will be described in the next section.
Immunohistochemical localization of 3-NT, DNP, and 4-HNE
For immunohistochemistry, 4 μm sections were deparaffined with xylol and rehydrated with ethanol. Endogenous peroxidase was quenched/inhibited with 4.5% H2O2 in methanol by incubation for 1.5 h at room temperature. The sections used for DNP immunohistochemistry were incubated with 0.2% DNPH in 2 N HCl for 60 min at room temperature in absence of light and then were extensively washed. Nonspecific adsorption was minimized by leaving the sections in 3% bovine serum albumin in phosphate buffer saline for 30 min. Sections were incubated overnight with a 1:700 dilution of anti-3-NT antibody  or with a 1:500 dilution of anti-DNP antibody or with a 1:100 dilution of anti-4-HNE antibody. After extensive washing with phosphate buffer saline, the sections were incubated with a 1:500 dilution of a peroxidase conjugated anti-rabbit Ig antibody (for 3-NT) or with a 1:500 dilution of a peroxidase conjugated anti-goat Ig or anti-mouse IgG (for DNP and 4-HNE, respectively) for 1 h, and finally incubated with hydrogen peroxide-diaminobenzidine for 1 min. Sections were counterstained with hematoxilin (for 3-NT and 4-HNE) or with methyl green (for DNP) and observed under light microscopy. All the sections from the four studied groups were incubated under the same conditions with the same antibodies concentration, and in the same running, so the immunostaining was comparable among the different experimental groups.
Antioxidant enzymes in renal cortex
Total SOD activity was assayed by a previously reported method using nitroblue tetrazolium (NBT) . To measure Mn-SOD activity, Cu,Zn-SOD was inhibited with diethyldithiocarbamic acid (DDC) . Cu,Zn-SOD activity was obtained by subtracting the activity of the DDC-treated samples from that of total SOD activity. GPx activity was assayed as previously described  and GR activity was assayed measuring the disappearance of NADPH . CAT activity was assayed by a method based on the disappearance of H2O2 .
Results are expressed as the mean ± SEM. Data were analyzed by one-way ANOVA followed by Bonferroni's multiple comparisons. Non-paired t-test was used to compare the quantitative histological damage data using the software Prism 3.02 (GraphPad, San Diego, CA, USA). P ≤ 0.05 was considered statistically significant.
This work was supported by CONACYT (#40009-M). SAMC was a generous gift of Wakunaga Pharmaceutical Co., Ltd. (Hiroshima, Japan).
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