Protective effects of garlic powder against potassium dichromate-induced oxidative stress and nephrotoxicity

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Food and Chemical Toxicology 46 (2008) 619–627

Protective effects of garlic powder against potassiumdichromate-induced oxidative stress and nephrotoxicity

Jose Pedraza-Chaverri a,*, Paola Yam-Canul a, Yolanda I Chirino a,Dolores Javier Sanchez-Gonzalez b, Claudia Marıa Martınez-Martınez b,

Cristino Cruz c, Omar N Medina-Campos a

a Facultad de Quımica, Departamento de Biologıa, Edificio F, Segundo Piso, Laboratorio 209, Universidad Nacional Autonoma de Mexico (UNAM),

Ciudad Universitaria, 04510 D.F., Mexicob Departamento de Biologıa Celular, Escuela Medico Militar, Universidad del Ejercito y Fuerza Aerea, Cerrada de Palomas y Batalla de Celaya,

Col. Lomas de San Isidro, Delegacion Miguel Hidalgo, 11200 D.F., Mexicoc Departamento de Nefrologıa, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Delegacion Tlalpan, 14000 D.F., Mexico

Received 28 February 2007; accepted 9 September 2007

Abstract

Potassium dichromate (K2Cr2O7)-induced nephrotoxicity is associated with oxidative stress. In the present work the effect of garlicpowder, a recognized antioxidant, on K2Cr2O7-induced nephrotoxicity and oxidative stress was studied. Rats were fed a 2% garlic pow-der diet for 1 month. A single injection of K2Cr2O7 (15 mg/kg) to rats induced tubule interstitial damage and an increase in the followingmarkers of renal injury 2 days later: blood urea nitrogen (4.6-fold), serum creatinine (9.7-fold), proteinuria (35.9-fold), urinary excretionof N-acetyl-b-D-glucosaminidase (12.9-fold) and glutathione-S-transferase (2.3-fold) and a decrease of 65% in serum glutathione perox-idase activity. In addition, K2Cr2O7 injection increased the following nitrosative and oxidative stress markers in kidney: 3-nitrotyrosine(1.9-fold), 4-hydroxy-2-nonenal (2.1-fold), malondialdehyde (1.8-fold) and protein carbonyl content (1.7-fold). It was found that garlicpowder feeding was able to prevent by 44–71% the alterations in the markers of renal injury studied, by 55% the histological damage, andby 47–100% the increase in markers of oxidative and nitrosative stress. It is concluded that the ability of garlic powder to ameliorateK2Cr2O7-induced renal injury is associated with its antioxidant properties. Our data support the use of garlic powder as a renoprotectiveagent.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Garlic; Potassium dichromate; 3-Nitrotyrosine; 4-Hydroxy-2-nonenal; Malondialdehyde; Protein carbonyl content; Nephrotoxicity; Antiox-idant; Oxidative stress

1. Introduction

Potassium dichromate (K2Cr2O7) is a chemical com-pound widely used in metallurgy, chrome plating, chemicalindustry, textile manufacture, wood preservation, photog-raphy and photoengraving, refractory and stainless steelindustries and cooling systems (Barceloux, 1999). The oxi-dation state and solubility of chromium (Cr) compounds

0278-6915/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.fct.2007.09.088

* Corresponding author. Tel./fax: +52 55 5622 3878.E-mail address: [email protected] (J. Pedraza-Chaverri).

determine their toxicity. In contrast to Cr(III), which is anaturally occurring form and an essential trace elementfor humans and others mammals, Cr(VI) compounds arehighly toxic (Wang et al., 2006). K2Cr2O7 is a hexavalentform of Cr and has been demonstrated to induce oxidativestress and carcinogenic in nature (Stohs and Bagchi, 1995;Norseth, 1981; Von Burg and Liu, 1993; Bagchi et al.,2002). The kidney is the principal route of Cr excretionand it has been reported that acute exposure induces anincrease in Cr kidney content on K2Cr2O7-treated rats(Pedraza-Chaverri et al., 2005). Exposition to Cr(VI)

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produced anatomical lesions at the level of the proximaltubular cells (Franchini et al., 1978) and lipid peroxidationin human kidney (Huang et al., 1999). Interestingly, evi-dences suggest that reactive oxygen species (ROS) areinvolved in Cr(VI)-induced cell injury (Sengupta et al.,1992; Liu and Shi, 2001; Stohs and Bagchi, 1995; Bagchiet al., 2002; Travacio et al., 2001). Cr reduction intermedi-ates [Cr(V) and Cr(IV)], may be toxic as they involve ROSproduction (Stohs et al., 2000; Shi and Dalal, 1990,1994;OBrien and Kortenkamp, 1994) which may be generatedduring physiological conditions. In vitro, chromate reduc-tion via hydrogen peroxide (H2O2) has been shown to pro-duce hydroxyl radical (OH�) via a Fenton-like reaction(OBrien and Kortenkamp, 1994; Aiyar et al., 1991; Shiand Dalal, 1990; Liu et al., 1997; Tsou et al., 1996). Inin vivo experiments have been shown that K2Cr2O7 exposi-tion induces oxidative and nitrosative stress measured asprotein carbonyl content and 3-nitrotyrosine (3-NT)immunostaining (Barrera et al., 2003a,b; Pedraza-Chaverriet al., 2005). The role of oxidative stress in the renal dam-age induced by K2Cr2O7 has been supported by the factthat some antioxidants such as a-tocopherol, ascorbic acid,and glutathione (GSH) (Appenroth and Winnefeld, 1998;Arreola-Mendoza et al., 2006; Na et al., 1992; Sugiyama,1992; Hojo and Satomi, 1991; Standeven and Wetterhahn,1991) and the previous induction of heme oxygenase-1(Barrera et al., 2003a,b) are able to ameliorate K2Cr2O7-induced nephrotoxicity and oxidative damage.

To our knowledge, the potential protective effect ofgarlic powder on K2Cr2O7-induced nephrotoxicity hasnot been explored. Garlic is a particularly rich source oforganosulfur compounds which are responsible for its fla-vor and aroma, as well as for its potential health benefits(Lawson, 1996, 1998; Reuter et al., 1996). c-Glutamyl-S-alkyl-L-cysteines and S-alkyl-L-cysteine sulfoxides arefound mainly in raw garlic cloves (Lawson, 1996). Themost abundant organosulfur compound in raw garliccloves is alliin (S-allylcysteine sulfoxide), which is presentat 10 mg/g fresh garlic (Lawson, 1998). When garlic clovesare cut or when the powder of dried cloves becomes wet ina non-acid solution, the cysteine sulfoxides, which areodorless, are very rapidly converted to a new class of com-pounds, the thiosulfinates which are responsible for theodor of freshly chopped garlic. This is because cysteinesulfoxides, which are located only in the clove mesophyllstorage cells, come in contact with the enzyme allinase oralliin lyase, which is located only in the vascular bundlesheath cells. Due to the abundance of alliin, the mainthiosulfinate formed upon crushing garlic cloves is allicin(Lawson, 1996).

The antioxidant ability of garlic in several presentationsis well known (Banerjee et al., 2003a; Rahman and Lowe,2006) and has been associated with its protective effect inseveral experimental models (Thabrew et al., 2000; Pedr-aza-Chaverri et al., 2000a; Gedik et al., 2005; Ip et al.,1992; Liu et al., 1992; Pal et al., 2006; Reuter et al, 1996;Sener et al., 2005).

In fact, a protective effect of a diet with garlic powderhas been observed in cardiac ischemia and reperfusion(Rietz et al., 1993), adriamycin-induced toxicity (Thabrewet al., 2000), gentamicin-induced nephrotoxicity (Pedraza-Chaverri et al., 2000a), azoxymethane-induced damage(Khanum et al., 1998), and hypercholesterolemic (Heinleand Betz, 1994; Kempaiah and Srinivasan, 2004b; Gorin-stein et al., 2006; Durak et al., 2002) and high fat (Kempa-iah and Srinivasan, 2004a) diet-induced oxidative damage.In addition, the antioxidant properties of garlic extractshave been shown in vitro. Extracts of garlic powder are ableto inhibit Cu2+-induced low-density lipoprotein oxidation(Lewin and Popov, 1994; Pedraza-Chaverri et al., 2004)and to scavenge OH� (Lewin and Popov, 1994; Pedraza-Chaverri et al., 2006; Torok et al., 1994), superoxide anionðO��2 Þ (Pedraza-Chaverri et al., 2006), H2O2 (Pedraza-Chaverri et al., 2006), and peroxynitrite (ONOO�) (Pedr-aza-Chaverri et al., 2007). Based on the above informationwe made the hypothesis that garlic powder may reduceK2Cr2O7-induced renal injury. The aim of this study wasto examine the effect of a 2% garlic powder supplementeddiet on K2Cr2O7-induced nephrotoxicity and oxidativeand nitrosative stress.

2. Materials and methods

2.1. Reagents

Guanidine hydrochloride, p-nitrophenyl-N-acetyl-b-D-glucosaminide,2,4,-dinitrophenylhydrazine (DNPH), streptomycin sulfate, 1-methyl-2-phenylindole, tetramethoxypropane, 1-chloro-2,4-dinitrobenzene (CDNB),GSH, glutathione reductase (GR), and nicotine-adenine-dinucleotidephosphate (NADPH) were purchased from Sigma Chemical Co. (St.Louis, MO, USA). Trichloroacetic acid, HCl, H2O2, acetonitrile, andmethanol were purchased from Mallinckrodt Baker (Xalostoc, Mexico).Commercial kits for the measurement of blood urea nitrogen (BUN) andcreatinine levels (Sera-pak plus urea and Sera-pak plus creatinine) werefrom Bayer (Tarrytown, NY, USA). Mouse monoclonal anti-4-hydroxy-2-nonenal (4-HNE) antibodies (Cat. #24325) were from Oxis International,Inc. (Portland, OR, USA). Mouse monoclonal antibodies against 3-NT(Cat. #189542) were purchased from Cayman Chemical Co. (Ann Arbor,MI, USA). The secondary antibodies biotin SP conjugated AffiniPuredonkey anti-mouse IgG (Cat. #715-065-151) were purchased from Jack-son ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). Declerewas from Cell Marque (Hot Springs, AR, USA). ABC-kit Vectastain wasfrom Vector Laboratories (Orton Southgate, Peterborough, UK). Diam-inobenzidine substrate (Cat. #K3466) and Mayer’s Hematoxylin (Lillie’sModification) (Cat. #S3309) were from DAKO Corporation (Carpinteria,CA, USA). A commercial natural garlic powder (Code Number 91374,Expiration date May 9, 2008) manufactured by Tone Brothers Inc.(Ankeny, IA, USA) was used. The nutritional information of this par-ticular garlic powder is the following: calories: 0, calories from fat: 0, totalfat: 0 g, trans fat: 0 g, saturated fat: 0 g, cholesterol: 0 mg, sodium: 0 mg,total carbohydrate: 0 g, dietary sugars: 0 g, fiber: 0 g, and protein: 0 g.

2.1.1. H2O2 scavenging activity of garlic powder

In previous papers, we have shown that a garlic powder fromMcCormick has in vitro reactive oxygen and nitrogen species scavengingproperties (Pedraza-Chaverri et al., 2004, 2006, 2007). Therefore, with thepurpose to evaluate the antioxidant ability of the garlic powder used in thepresent study, we measured its in vitro H2O2 scavenging ability (expressedas IC50) as previously described (Pedraza-Chaverri et al., 2006). Thisresult was compared with that obtained from garlic powder from

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McCormick, which has been used in our previous studies (Pedraza-Chaverri et al., 1998, 2000a,b, 2001). We measured the IC50 for garlicpowder obtained from both commercial sources: Tones and McCormick.The IC50 calculated in our assay conditions was of 1.2 ± 0.1 mg/mL(n = 8) for the one obtained from Tones and of 1.3 ± 0.1 mg/mL (n = 9)for that obtained from McCormick (p = NS). These data suggest that theantioxidant ability of both garlic presentations is essentially similar. Wewere unable to characterize by HPLC the garlic powder used in the presentstudy because of the lack of appropriate standards.

2.2. Animal diet

Harlan Teklad Global diet 2018S sterilized (Harland Teklad, Madison,WI, USA) was used as a control diet. The standard diet consisted of crudeprotein 18.80%, crude oil 6%, crude fiber 3.8%, carbohydrate 57.26%,starch 41.19% and sugar 4.91%. The experimental rats were fed thestandard diet enriched with 2% garlic powder as previously described byRietz et al. (1993). Our previous studies with 2% garlic powder diet(Pedraza-Chaverri et al., 1998, 2000a,b, 2001) were also based in the workof Rietz et al. (1993).

2.3. Experimental design

Twenty female Wistar rats (200–230 g) were used. Experimental workfollowed the guidelines of Norma Official Mexicana Guide for the use andcare of laboratory animals (NOM-062-ZOO-1999) and for the disposal ofbiological residues (NOM-087-ECOL-1995). All animals were placed inmetabolic cages and randomly divided in four groups. The first (CT) andthird (K2Cr2O7) groups were fed with diet without garlic powder. Thesecond (CT + GA) and fourth (K2Cr2O7 + GA) groups were fed a dietenriched with 2% garlic powder (Rietz et al., 1993). All rats had free accessto water and food. After one month, the third and fourth groups receiveda single subcutaneous injection of K2Cr2O7 (15 mg/kg) (Pedraza-Chaverriet al., 1995; Barrera et al., 2003a,b) and urine was collected every 24 h for2 days. At the end of the study (48 h), rats were sacrificed by decapitationand blood was collected at room temperature to obtain serum. Bothkidneys were obtained to perform biochemical, histological and immu-nohistochemical analyses.

2.4. Renal function

K2Cr2O7-induced renal injury was evaluated by the following markers:serum creatinine concentration, BUN levels, and serum glutathione per-oxidase (GPx) activity, as well as urinary excretion of total protein, N-acetyl-b-D-glucosaminidase (NAG) and glutathione-S-transferase (GST)(Barrera et al., 2003a; Pedraza-Chaverri et al., 2000a; Badary et al., 2005).Serum creatinine concentration and BUN concentration were measuredwith an autoanalyzer (Technicon RA-1000, Bayer Tarrytown, NY, USA).Serum GPx activity was measured at 340 nm using GR and NADPH in acoupled reaction. One unit of GPx was defined as the amount of enzymethat oxidizes 1 lmol of NADPH/min and the data were expressed as U/mL. Total protein in urine was measured by a turbidimetric method with12.5% trichloroacetic acid at 420 nm (Barrera et al., 2003a) and the datawere expressed as mg/24 h. Urinary NAG activity was determined at405 nm using p-nitrophenyl-N-acetyl-b-D-glucosaminide as substrate andthe data were expressed as U/24 h (Pedraza-Chaverri et al., 2000a). Oneunit of NAG was defined as the amount of enzyme that releases 1 lmol ofp-nitrophenol in the assay conditions. GST (EC 2.5.1.18) are cytosolicenzymes involved in the binding and detoxification of toxic compounds.The urinary excretion of total GST (Badary et al., 2005; Liu et al., 2007;Peters et al., 1997; Bomhard et al., 1990) and a (proximal tubules) and p(distal and collecting tubules) GST classes (Green et al., 2005; Usudaet al.,1999; Kharasch et al., 1997) has been measured in several studies toevaluate the renal tubular damage in rats. Urinary total GST activity wasdetermined by the method of Habig et al. (1974). The reaction mixture(CNDB, GSH and urine) was incubated for 1 h at room temperature andthen the absorbance was measured at 340 nm. The GST activity is

expressed as nmol of GSH-CDNB conjugate formed/min/24 h. To verifythat we were measuring the urinary GST activity, a urinary sample wasboiled for 10 min and subsequently an aliquot of this sample was incu-bated with GSH and CDNB at room temperature for 60 min and theabsorbance was registered at 340 nm. In contrast with non-heated urinesamples, the absorbance remained unchanged along the incubation timeclearly indicating absence of enzyme activity.

2.5. Histological and immunohistochemical analyses

For light microscopy, kidney tissue was fixed by immersion inbuffered formalin (pH 7.4) and embedded in paraffin. For histologicalanalysis, sections (3 lm) were stained with hematoxylin and eosin. Thehistological profile of proximal tubules from 5 randomly selected fields (5rats per experimental group) was recorded using KS-300 software (CarlZeiss, Jena, Germany). The percentage of tubular area with histopa-thological alterations like swelling, cytoplasmic vacuolization, desqua-mation or necrosis was obtained. For immunohistochemistry, kidneysections (3 lm) were deparaffined and then boiled in Declere to unmaskantigen sites; the endogenous activity of peroxidase was quenched with0.03% H2O2 in absolute methanol. Kidney sections were incubatedovernight at 4 �C with 1:70 dilution of anti 3-NT and 1:200 dilution ofanti 4-HNE antibodies in phosphate buffered saline (PBS). Followingremoval of the primary antibodies and repetitive rinsing with PBS, slideswere incubated with a 1:500 dilution of biotinylated goat anti-IgG sec-ondary antibody. Bound antibodies were detected with avidin–biotinyl-ated peroxidase complex ABC-kit Vectastain and diaminobenzidinesubstrate. After appropriate washing in PBS, slides were counterstainedwith hematoxylin. All specimens were examined by light microscopy(Axiovert 200M, Carl Zeiss, Jena, Germany). For automated mor-phometry analysis, the percentage of positive cells (brown staining) wasdetermined with a computerized image analyzer KS-300 3.0 (Carl Zeiss,Jena, Germany). This equipment automatically detects positive cellsdetermining their percentage per field. Five random fields per kidneywere studied at 100 · magnification (total area 1 · 106 square microns)comparing the different groups. All sections were incubated under thesame conditions with the same concentration of antibodies and in thesame running, so the immunostaining was comparable among the dif-ferent experimental groups. For the negative control, preimmune goatserum was used instead of the primary antibodies (Sanchez-Gonzalezet al., 2004; Orozco-Ibarra et al., 2007).

2.6. Malondialdehyde (MDA) and protein carbonyl content

MDA in the kidney tissue was measured using a standard curve oftetramethoxypropane. A solution of 1-methyl-2-phenylindole in a mixtureof acetonitrile/methanol (3:1) was added to the renal homogenates and thereaction was started by adding 37% HCl. Optical density was measured586 nm after 1 h of incubation at 45 �C (Gerard-Monnier et al., 1998).Data were expressed as nmol MDA/mg protein. Protein carbonyl contentin the kidney tissue was determined by the method of Reznick and Packer(1994). The renal homogenates were incubated with streptomycin sulfateto remove nucleic acids overnight. Further renal homogenates were trea-ted with DNPH and HCl and finally with guanidine hydrochloride.Assessment of carbonyl formation was done on the basis of formation ofprotein hydrazone by reaction with DNPH. The absorbance was measuredat 370 nm. Protein carbonyl content was expressed as nmol of carbonyl/mg protein.

2.7. Statistical analyses

Data were expressed as mean ± S.E.M. Data were analyzed with thesoftware Prism 3.02 (GraphPad, San Diego, CA, USA) by one-wayanalysis of variance followed by Bonferroni multiple comparisons method.The quantification of the histological damage was compared by Mann–Whitney U test. The H2O2 scavenging ability was compared with a non-paired t test. A p value of p < 0.05 was considered significant.

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3. Results

Body weight was unchanged in all groups studied on day2 (Table 1). We first investigated whether the 2% garlic diet

Table 1Body weight and renal injury markers 48 h after vehicle or K2Cr2O7 injection

CT

Body weight, g (n = 5) 218Blood urea nitrogen, mg/dL (n = 5) 22.1Serum creatinine, mg/dL (n = 4–5) 0.37Proteinuria, mg/24 h (n = 4–5) 2.2Serum GPx activity, U/mL (n = 5) 0.91Urinary NAG, U/24 h (n = 3–5) 0.21Urinary GST, nmol CDNB conjugated formed/min/24 h (n = 5) 19.85

GPx: glutathione peroxidase; NAG: N-acetyl-p-D-glucosaminidase; GST: glutwith a standard diet; CT + GA: control fed with a 2% garlic powder diet, K2Cfed with a 2% garlic powder diet and injected with a single dose of K2Cr2O7.

a p < 0.001.b p < 0.05 vs. CT.c p < 0.05.d p < 0.001.e p < 0.001 vs. K2Cr2O7.

Fig. 1. Structural and immunohistochemical analysis from kidney sections (3hematoxylin and eosin staining (a–d). Slices from control non-treated group (normal architecture. Slices from K2Cr2O7-treated rats (c) showed extensive dam(arrowheads) and tubular casts (arrow). Slices from K2Cr2O7-treated rats fed awith few epithelial tubular cells affected. Kidney sections from control non-tre(f and g) show negative 3-NT and 4-HNE immunostaining, respectively. In4-HNE (k) immunostaining. Kidney sections from K2Cr2O7-treated rats fed4-HNE (l) immunostaining 100·.

reduces or prevents renal dysfunction and structural injuryinduced by K2Cr2O7 administration. As shown in Table 1,after 48 h of a single K2Cr2O7 injection, the rats presenteda marked reduction of renal function compared to CT

CT + GA K2Cr2O7 K2Cr2O7 + GA

± 6.5 216.1 ± 3.2 219.7 ± 5.5 217.9 ± 6.9± 1.2 27.7 ± 2.3 101.1 ± 7.4a 66.7 ± 7.5a,c

± 0.08 0.35 ± 0.03 3.6 ± 0.29a 1.81 ± 0.47c

± 0.5 3.1 ± 0.8 79.1 ± 2.6a 34.0 ± 1.9a,d

± 0.03 0.97 ± 0.08 0.32 ± 0.01a 0.64 ± 0.03a,d

± 0.04 0.22 ± 0.02 2.7 ± 0.21a 1.22 ± 0.20b,d

± 3.1 19.26 ± 3.5 45.41 ± 2.8a 27.42 ± 3.0e

athione transferase; CDNB: 1-chloro-2,4-dinitro-benzene; CT: control fedr2O7 group injected with a single dose of K2Cr2O7, K2Cr2O7 + GA groupData are mean ± SEM.

lm) from all studied groups. Histological evaluation was performed usinga) and group fed a diet supplemented with 2% garlic powder (b) showedage and most of cortical tubules showed necrosis (asterisks), vacuolizationdiet supplemented with 2% garlic powder (d) showed lesser tissue damage

ated rats (e and i) and rats fed a diet supplemented with 2% garlic powdercontrast, slices from K2Cr2O7 treated rats showed positive 3-NT (g) anda diet supplemented with 2% garlic powder showed lesser 3-NT (h) and

Table 2Quantitative data of the immunohistochemistry studies (area, %)

CT GA K2Cr2O7 K2Cr2O7+GA

3-NT 9.02 ± 0.64 7.5 ± 0.77 16.88 ± 0.99a 8.79 ± 0.97b

4-HNE 5.45 ± 0.66 4.88 ± 0.61 11.79 ± 0.83a 7.12 ± 0.52b

CT: control fed with a standard diet; GA: control fed with a 2% garlicpowder diet, K2Cr2O7 group injected with a single dose of K2Cr2O7,K2Cr2O7 + GA group fed with a 2% garlic powder diet and injected with asingle dose of K2Cr2O7. 3-NT: 3-nitrotyrosine; 4-HNE: 4-hydroxy-none-nal. Data are mean ± SEM, n = 5.

a p < 0.001 vs. CT.b p < 0.001 vs. K2Cr2O7.

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group that was characterized by a significant increase inBUN (4.6-fold), and in serum creatinine concentration(9.7-fold) and in urinary excretion of total protein(35.9-fold), NAG (12.9-fold) and GST (2.3-fold) and by asignificative decrease of 65% in serum GPx activity. The2% garlic diet attenuated the increase in BUN (by 44%),in serum creatinine concentration (by 55%) and in urinaryexcretion of total protein (by 71%), NAG (by 62%), andGST (by 69%) and the decrease in serum GPx activity(by 48%) (Fig. 2b). These data suggest that 2% garlic pow-der diet has a renoprotective effect in this experimentalmodel; thus, we decided to further investigate whether thisantioxidant confers histological protection. We found thatgarlic powder diet was also able to ameliorate by 55% thepercentage of area with histological damage in K2Cr2O7-treated rats (32.58 ± 2.2 in K2Cr2O7 + GA group vs.72.50 ± 3.3% in K2Cr2O7 group, n = 5, p < 0.01) (Fig. 1).Slices from CT group showed normal architecture. Slicesfrom K2Cr2O7 + GA treated rats had lesser tissue damagewith few epithelial tubular cells affected (Fig. 1). Thus, ourfindings clearly show the 2% garlic diet ameliorates theK2Cr2O7-induced nephropathy.

Taking into account the previous data and the antioxi-dant and ROS scavenging properties of garlic powder, we

Fig. 2. Renal content of (a) malondialdehyde (MDA) (n = 5) and (b)protein carbonyl (n = 5) in the groups of rats studied: (1) CT, (2) GA, (3)K2Cr2O7, and (4) K2Cr2O7 + GA. Rats were studied 2 days after K2Cr2O7

injection (15 mg/kg). Two percent garlic powder was given in the food 1month before and 2 days after K2Cr2O7 injection. ap < 0.01 vs. CT,bp < 0.01 vs. K2Cr2O7. cp < 0.001 vs. CT, dp < 0.05 vs. K2Cr2O7.

decided to analyze whether the renoprotective effect of thisgarlic diet was related with reduction of oxidative andnitrosative stress in K2Cr2O7-treated rats which wereevaluated by renal immunohistochemistry for 4-HNE and3-NT, respectively. As shown in Fig. 1, a negative immuno-staining for 4-HNE and 3-NT was observed in cortexsections of CT group. In contrast, a strong immunostainingfor 3-NT (1.9-fold) and 4-HNE (2.1-fold) was observed inrenal cortex from K2Cr2O7-treated rats (Fig. 1, Table 2).Garlic diet protected the kidney of nitrosative and oxida-tive stress that was evinced by a weak immunoreactivityof 3-NT and 4-HNE in renal cortex from K2Cr2O7 + GAgroup (Fig. 1) which was confirmed by quantitative data(Table 2). The percentage of protection with garlic was of100% and 79% for 3-NT and 4-HNE, respectively. Further-more, the renal content of MDA and protein carbonyl wasmeasured as additional marker of oxidative stress. Asshown in Fig. 2, after 48 h of a single K2Cr2O7 injection,both MDA and protein carbonyl content increased by79% and 47%, respectively. The 2% garlic diet attenuatedthe increase in renal content of MDA (by 79%) and in pro-tein carbonyl (by 47%). Thus our data suggest a clear cor-relation between the renoprotective effects of 2% garlic dietwith the amelioration of oxidative and nitrosative stress.

4. Discussion

Our data clearly show that K2Cr2O7-induced renalinjury and oxidative stress were significantly amelioratedin 2% garlic powder-fed rats which confirm that the garlicpowder used in this study has in vivo antioxidant proper-ties. In fact, we showed that the in vitro H2O2 scavengingability of this garlic presentation is similar to that one usedin previous studies (Pedraza-Chaverri et al., 1998, 2000a,b,2001). Using a diet with 2% garlic powder from the samecommercial brand used in this study, it was found thathypertension is significantly ameliorated in rats (Pedraza-Chaverri et al., 1998). In addition, we have observed a cleardecrease in (a) renal and hepatic H2O2 production in nor-mal rats (Pedraza-Chaverri et al., 2001), (b) hyperlipidemiain rats with chronic aminonucleoside nephrosis (Pedraza-Chaverri et al., 2000b), and (c) nephrotoxicity and oxida-tive stress induced by gentamicin in rats (Pedraza-Chaverriet al., 2000a) with a 2% garlic diet obtained from a different

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commercial brand (McCormick). The components of thegarlic powder used in this work were not quantified, butthe composition of garlic powder has been largely studiedand characterized (Lawson, 1996). Although the specificconditions used to prepare the commercial garlic powderused in this study are not known, usually, the garlic powderis prepared dehydrating garlic cloves at low oven tempera-tures (50–60 �C) and then pulverized. In addition, the com-position of the garlic powder for spices has beendetermined and is known that the sulfur content of garlicpowder is 3% and that the main organsulfur compoundsare alliin (10–17 mg/g) and c-glutamylcysteines (12–35 mg/g) (Lawson, 1998). Our data are relevant taking intoaccount that occupational exposure to Cr has been associ-ated with acute renal failure (Sharma and Singhal, 1978;Picaud et al., 1991; Franchini et al., 1978).

This protective effect is consistent with the beneficialeffect of feeding 2% garlic powder observed in severalexperimental models (Rietz et al., 1993; Thabrew et al.,2000; Pedraza-Chaverri et al., 2000a,b; Kempaiah andSrinivasan, 2004a,b; Durak et al., 2002; Ip et al., 1992;Schaffer et al., 1997; Liu et al., 1992) and with thein vitro ROS scavenging activity of garlic powder (Pedr-aza-Chaverri et al., 2006, 2007). Furthermore, garlicextracts are able to confer protection against oxidativedamage. Banerjee et al. (2003b) found that the administra-tion of raw garlic homogenate orally for 30 days preventedisoproterenol-induced myocardial necrosis and oxidativestress in rats. In addition, Sener et al. (2005) found a pro-tective effect of aqueous garlic extract on ischemia/reperfu-sion induced hepatic injury and oxidative stress and Palet al. (2006) found that fresh garlic homogenate protectedrats against isoniazid and rifampicin-induced hepatic dam-age and oxidative stress. Furthermore, aqueous garlicextract ameliorated liver fibrosis and oxidative damageinduced by biliary obstruction in rats (Gedik et al., 2005).

K2Cr2O7-induced renal injury has been associated withenhanced 3-NT immunostaining suggesting that ONOO�,a strong oxidant and nitrating agent, is involved in therenal damage (Barrera et al., 2003a). This observationwas confirmed in the present study. The ability of the 2%garlic powder diet to prevent 3-NT immunostaining maybe related to the ONOO� scavenging capacity of extractsof garlic powder (Pedraza-Chaverri et al., 2007). 3-NT isthought to be a relatively specific marker of oxidative dam-age mediated by ONOO�, which is produced by the reac-tion between O��2 and nitric oxide (NO�) (Oldreive andRice-Evans, 2001). The increase in 3-NT production maybe secondary to the increase of either O��2 or NO� and ithas been documented that K2Cr2O7 enhances O��2 produc-tion (Liu and Shi, 2001; Stohs and Bagchi, 1995; Pritchardet al., 2000; Sugiyama, 1992). The O��2 (Pedraza-Chaverriet al., 2006), OH� (Lewin and Popov, 1994; Pedraza-Chav-erri et al., 2004) and H2O2 (Pedraza-Chaverri et al., 2006)scavenging ability of extracts of garlic powder also maybe involved in this protective effect. Alliin, the main S-alkyl-L-cysteine sulfoxide present in garlic cloves and garlic

powder, and allicin, produced by the enzymatic action ofallinase, and the compounds derived from its transforma-tion, may be responsible, at least in part, of the protectiveeffect of a diet with 2% garlic powder on K2Cr2O7-inducedrenal injury and oxidative stress. In fact, the antioxidantproperties of alliin and allicin have been largely studied.Sangeetha and Quine (2006) found that alliin was able toameliorate the isoproterenol-induced (a) cardiac damage,(b) lipid peroxidation and (c) the decrease in GSH, vita-mins C and E levels and GR and GST activities. Augustiand Sheela (1996) found that alliin ameliorates the diabeticcondition of alloxan treated rats, which was associatedwith an increase in GSH levels and decrease in lipid perox-idation in heart, kidney and liver, and increase of superox-ide dismutase (SOD) and catalase (CAT) activities in liver.Helen et al. (2003) found that alliin ameliorated the lipidperoxidation and prevents the decrease in CAT and SODactivities and in vitamins A, C and E levels induced by nic-otine in heart, liver and lung of rats. In addition, it hasbeen found in in vitro studies that alliin is able to scavengeOH� (Kourounakis and Rekka, 1991), O��2 (Chung, 2006)and H2O2 (Ide et al., 1996) and to inhibit lipid peroxidation(Ide et al., 1996). Furthermore, oral pretreatment of allicinprevented D-galactosamine-induced hepatitis, the decreasein the activities and levels of the antioxidant enzymesSOD, CAT, GPx and GST, the reduction in GSH levels,and the increase in lipid peroxidation in liver (Vimal andDevaki, 2004). In addition, it is known that allicin scav-enges OH� and prevents lipid peroxidation (Prasad et al.,1995), scavenges peroxyl radical, and inhibits methyl lino-leate oxidation (Okada et al., 2005). The ONOO� scaveng-ing ability of alliin and allicin remains to be studied. Inaddition, it is possible that the most abundant compoundsderived from garlic thiosulfinates (mainly allicin) in pres-ence of water (diallyl trisulfide, diallyl disulfide, and allylmethyl trisulfide) (Lawson, 1998), may be involved in theprotective effect of garlic powder diet on K2Cr2O7-inducednephrotoxicity. It is expected that some of these com-pounds be produced after garlic powder consumption tak-ing into account that allicin is formed by the enzymaticaction of allinase. In fact, it has been shown that diallyldisulfide and diallyl trisulfide have antioxidant propertiesboth in vivo (Fukao et al., 2004; Pedraza-Chaverri et al.,2003) and in vitro (Liu et al., 2006; Kim et al., 2005) andinduce detoxifying enzymes (Chen et al., 2004; Fukaoet al., 2004; Tsai et al., 2007).

In summary, our data show that the ability of a diet with2% garlic powder to ameliorate K2Cr2O7-induced renalinjury is associated with its antioxidant and ROS scaveng-ing properties. Our data support the use of garlic powderas a renoprotective agent.

Acknowledgements

This work was supported by CONACYT (Grant No.40009M). Axiovert 200 M confocal microscope was do-nated by Fundacion Gonzalo Rio Arronte IAP Mexico.

J. Pedraza-Chaverri et al. / Food and Chemical Toxicology 46 (2008) 619–627 625

We thank Giovanna Merchand-Reyes for her technicalsupport.

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