Nordihydroguaiaretic acid attenuates potassium dichromate-induced oxidative stress and nephrotoxicity

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Food and Chemical Toxicology 46 (2008) 1089–1096 www.elsevier.com/locate/foodchemtox

Nordihydroguaiaretic acid attenuates potassium dichromate-induced oxidative stress and nephrotoxicity Paola Yam-Canul a, Yolanda I Chirino a, Dolores Javier Sa´nchez-Gonza´lez b, Claudia Marı´a Martı´nez-Martı´nez b, Cristino Cruz c, Cleva Villanueva d, Jose´ Pedraza-Chaverri a,* a

Facultad de Quı´mica, Departamento de Biologı´a, Edificio F, Segundo Piso, Laboratorio 209, Universidad Nacional Auto´noma de Me´xico (UNAM), Ciudad Universitaria, 04510 D.F. Me´xico, Mexico b Departamento de Biologı´a Celular, Escuela Me´dico Militar, Universidad del Eje´rcito y Fuerza Ae´rea, Cerrada de Palomas y Batalla de Celaya, Col. Lomas de San Isidro, Delegacio´n Miguel Hidalgo, 11200 D.F. Me´xico, Mexico c Departamento de Nefrologı´a, Instituto Nacional de Ciencias Me´dicas y Nutricio´n Salvador Zubira´n, 14000 D.F. Me´xico, Mexico d Escuela Superior de Medicina del Instituto Politecnico Nacional, Plan de San Luis y Salvador Dı´az Miro´n S/N, Col. Casco de Santo Tomas, 11340 D.F. Me´xico, Mexico Received 13 May 2007; accepted 6 November 2007

Abstract Larrea tridentata also known as Creosote bush, Larrea, chaparral, greasewood or gobernadora has been used in the folk medicine for the treatment of several illnesses. The primary product that is present at high concentrations in the leaves from this plant is nordihydroguaiaretic acid (NDGA) which is a powerful antioxidant. On the other hand, potassium dichromate (K2Cr2O7)-induced nephrotoxicity is associated with oxidative stress. The aim of this work was to study the effect of NDGA on K2Cr2O7-induced nephrotoxicity and oxidative stress. Nephrotoxicity was induced by a single injection of K2Cr2O7 (15 mg/Kg). A group of K2Cr2O7-treated rats was administered NDGA by mini osmotic pumps (17 mg/Kg/day). The results show that NDGA was able to ameliorate the structural and functional renal damage evaluated by histopathological analysis and by measuring proteinuria, urinary excretion of N-acetyl-bD-glucosaminidase, serum creatinine, and serum glutathione peroxidase activity. In addition, immunostaining of 4-hydroxy-2-nonenal and 3-nitrotyrosine, markers of oxidative and nitrosative stress, respectively, was ameliorated by the NDGA treatment. These data strongly suggest that the antioxidant properties of NDGA are involved in its renoprotective effect in K2Cr2O7-treated rats. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Larrea tridentata; Antioxidant; Nephrotoxicity; Oxidative stress; Potassium dichromate

1. Introduction Larrea tridentata belongs to the family Zygophyllaceae and also is known as Creosote bush, Larrea, chaparral, greasewood or gobernadora, which dominates some areas of the desert southwest in the United States and Northern Mexico, as well as some areas of Argentina (Arteaga et al., 2005). Chaparral tea has been used in the folk medicine for *

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

0278-6915/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.11.003

the treatment of more than 50 ailments including rheumatism, arthritis, diabetes, gallbladder and kidney stones, and inflammation (Arteaga et al., 2005). This tea has antioxidant properties (Zang et al., 1999). In addition, L. tridentata is a notable source of natural products with approximately 50% of the leaves dry weight as extractable matter. The resin that covers the leaves yields 19 flavonoid aglycones, as well as several lignans, notably including nordihydroguaiaretic acid (4-[4-(3,4-dihydroxyphenyl)2,3-dimethylbutyl]benzene-1,2-diol, NDGA) (Arteaga et al., 2005). NDGA is a recognized antioxidant and more

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recently, it has been demonstrated that NDGA is a potent in vitro scavenger of peroxynitrite (ONOO), singlet oxygen, hydroxyl radical (OH), superoxide anion ðO 2 Þ, and hypochlorous acid (Floriano-Sanchez et al., 2006). The antioxidant effect of NDGA has been observed in renal and hepatic toxicity induced by ferric-nitrilotriacetate (Ansar et al., 1999), ozone induced tyrosine nitration in lungs (Floriano-Sanchez et al., 2006), and streptozotocininduced diabetic nephropathy (Anjaneyulu and Chopra, 2004). Ansar et al. (1999) found that NDGA was able to prevent the increase in renal and hepatic lipid peroxidation and H2O2 generation and the decrease in renal and hepatic glutathione (GSH) content and GSH-S-transferase, GSH reductase, glucose-6-phosphate dehydrogenase and catalase activities induced by ferric-nitrilotriacetate. Furthermore, NDGA treatment was able to prevent ozoneinduced tyrosine nitration in lungs (Floriano-Sanchez et al., 2006). In addition, Anjaneyulu and Chopra (2004) found that NDGA prevented the increase in renal malondialdehyde levels and the decrease in renal GSH content and in superoxide dismutase and catalase activities induced by streptozotocin in rats. Furthermore, it has been found that NDGA has several health beneficial properties including: (a) inhibition of the growth of several human cancer types both in vitro and in vivo (Huang et al., 2004; Hofmanova et al., 2002), (b) chemopreventive ability in models of carcinogenesis (Moody et al., 1998; Ansar et al., 1999), (c) degradation of preformed Alzheimer’s b-amyloid fibrils in vitro (Ono et al., 2002), and (d) protection of cultured rat hippocampal neurons against the toxicity of amyloid b-peptide (Goodman et al., 1994), interrupting a neurodegenerative pathway relevant to the pathophysiology of Alzheimer’s disease. On the other hand, potassium dichromate (K2Cr2O7) is a chemical compound widely used in metallurgy, chrome plating, chemical industry, textile manufacture, wood preservation, photography and photoengraving, refractory and stainless steel industries and cooling systems (Barceloux, 1999). The oxidation state and solubility of chromium compounds determine their toxicity. In contrast to Cr3+, which is a naturally occurring form and an essential trace element for humans and other mammals, Cr6+ compounds are highly toxic (Wang et al., 2006). K2Cr2O7 is a hexavalent form of Cr and has been demonstrated to induce oxidative stress 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 chromium excretion and it has been reported that acute exposure induces an increase in chromium kidney content on K2Cr2O7-treated rats (PedrazaChaverri et al., 2005). Exposition to Cr6+ produced anatomical lesions at the level of the proximal tubular cells (Franchini et al., 1978) and lipid peroxidation in human kidney (Huang et al., 1999). Interestingly, evidences suggest that reactive oxygen species (ROS) are involved in Cr6+-induced cell injury (Liu and Shi, 2001; Bagchi et al., 2002; Travacio et al., 2001). Chromium reduction intermediates (Cr5+ and Cr4+) may be toxic as they involve ROS

production (Stohs et al., 2000; Shi and Dalal, 1990, 1994), which may be generated during physiological conditions. In vitro, hydrogen peroxide (H2O2)-induced Cr6+ reduction has been shown to produce OH via a Fentonlike reaction (Aiyar et al., 1991; Shi and Dalal, 1990; Tsou et al., 1996). In vivo experiments have shown that K2Cr2O7 exposition induces renal oxidative and nitrosative stress measured as protein carbonyl content and 3-nitrotyrosine (3-NT) immunostaining, respectively (Barrera et al., 2003; Pedraza-Chaverri et al., 2005). The role of oxidative stress in the renal damage induced by K2Cr2O7 has been supported additionally by the fact that some antioxidants such as a-tocopherol, ascorbic acid, and GSH (Appenroth and Winnefeld, 1998; Arreola-Mendoza et al., 2006; Na et al., 1992; Sugiyama, 1992; Hojo and Satomi, 1991) and the previous induction of heme oxygenase-1 (HO-1) (Barrera et al., 2003) are able to ameliorate K2Cr2O7-induced nephrotoxicity and oxidative damage. To our knowledge, the potential protective effect of NDGA on K2Cr2O7-induced nephrotoxicity has not been explored. Based on the above information, the hypothesis was made that NDGA may reduce K2Cr2O7-induced renal injury. The aim of this study was to examine the effect of NDGA on K2Cr2O7-induced nephrotoxicity and oxidative and nitrosative stress. 2. Materials and methods 2.1. Reagents and materials Dimethyl sulfoxide (DMSO), p-nitrophenyl-N-acetyl-b-D-glucosaminide, NDGA, NADPH, GSH, and GSH reductase were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Trichloroacetic acid was purchased from Mallinckrodt Baker Inc. (Phillipsburg, NJ, USA). Commercial kits for the measurement of creatinine concentration (Sera-pak plus creatinine) were from Bayer (Tarrytown, NY, USA). Mouse monoclonal anti-4-hydroxy-2-nonenal (4-HNE) antibodies (Cat. # 24325) were purchased from Oxis International, Inc. (Portland, OR, USA). Mouse monoclonal antibodies anti-3-NT (Cat. # 189542) were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). The secondary antibodies biotin SP-conjugated AffiniPure donkey anti-mouse IgG (Cat. # 715-065151) were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). H2O2 was purchased from Mallinckrodt Baker (Xalostoc, Mexico). Declere was from Cell Marque (Hot Springs, AR, USA). ABC-kit Vectastain was from Vector Laboratories (Orton Southgate, Peterborough, UK). Diaminobenzidine substrate (Cat. # K3466) and Mayer’s Hematoxylin (Lillie’s Modification) (Cat. # S3309) were from DAKO Corporation (Carpinteria, CA, USA). Sodium pentobarbital was from Pzifer (Mexico). All other chemicals were reagent grade and commercially available. Alzet miniosmotic pumps Model 2004 (mean fill volume of 200 lL and mean pumping rate of 0.25 lL/h) were from Durect Corporation (Cupertino, CA, USA). Stainless steel metabolic cages were from Allentown Caging Equipment Co. (Allentown, NJ, USA).

2.2. Experimental design Experimental work followed the guidelines of Norma Official Mexicana Guide for the use and care of laboratory animals (NOM-062-ZOO1999) and for the disposal of biological residues (NOM-087-ECOL-1995). Fifty two female Wistar rats bred-in house (200–230 g) were used. Animals were provided with a standard commercial rat chow diet (Harlan Teklad Global diet 2018S sterilized, Harland Teklad, Madison, WI, USA) and water ad libitum. Housing room was maintained under constant condi-

P. Yam-Canul et al. / Food and Chemical Toxicology 46 (2008) 1089–1096 tions of temperature (21 ± 1°C), relative humidity (50–60%) and lighting (12-h light/dark cycle). The air was filtered until 5 lm particles and was exchanged 18 times/h; water was filtered by reverse osmosis. All animals were placed in individual stainless steel metabolic cages and randomly divided in four groups: control (CT), K2Cr2O7, K2Cr2O7 + NDGA, and K2Cr2O7 + DMSO. Rats from CT group (n = 11) received s.c. saline solution 0.9% (0.2 mL); rats from K2Cr2O7 group (n = 16) received s.c. K2Cr2O7 (15 mg/Kg) in a total volume of 0.2 mL (Barrera et al., 2003); rats from K2Cr2O7 + NDGA group (n = 15) received the same dose of K2Cr2O7 and NDGA (17 mg/Kg/day) delivered by mini-osmotic pumps (Floriano-Sanchez et al., 2006) 24 h before K2Cr2O7 injection and 2 days after K2Cr2O7 injection. NDGA was dissolved in DMSO (116 mg/200 ll). The dose of NDGA was very close to that used in a previous paper (20 mg/Kg) in which it was observed protective effect of NDGA in rats against ozone-oxidative damage (Floriano-Sanchez et al., 2006). Rats from K2Cr2O7 + DMSO group (n = 10) received the same dose of K2Cr2O7 and DMSO (33 mg/Kg/day) was delivered by mini-osmotic pumps 24 h before K2Cr2O7 injection. At the end of the study (48 h), rats were killed by decapitation and blood was collected at room temperature to obtain serum for biochemical determinations. Urine was collected for the last 24 h of the study. In addition, both kidneys were obtained to perform histological and immunohistochemical analyses. Rats were studied 48 h after K2Cr2O7 injection taking into account that at this time point is attained the highest renal damage (Pedraza-Chaverri et al., 2005).

2.3. Renal function K2Cr2O7-induced renal injury was evaluated by the following markers: serum creatinine concentration, serum glutathione peroxidase (GPx) activity, and urinary excretion of total protein and N-acetyl-b-D-glucosaminidase (NAG) (Barrera et al., 2003). Serum creatinine concentration was measured with an autoanalyzer (Technicon RA-1000, Bayer Tarrytown, NY, USA). Serum GPx activity was measured in serum at 340 nm using GSH reductase and NADPH in a coupled reaction (PedrazaChaverri et al., 2005). One unit of GPx was defined as the amount of enzyme that oxidizes 1 lmol of NADPH/min and the data were expressed as U/mL. Total protein in urine was measured by a turbidimetric method with 12.5% trichloroacetic acid at 420 nm (Barrera et al., 2003) and the data were expressed as mg/24 h. Urinary NAG activity was determined at 405 nm using p-nitrophenyl-N-acetyl-b-D-glucosaminide as substrate and the data were expressed as U/24 h (Pedraza-Chaverri et al., 2005). One unit of NAG was defined as the amount of enzyme that releases 1 lmol of p-nitrophenol in the assay conditions.

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negative control, preimmune goat serum was used instead of the primary antibodies (Sanchez-Gonzalez et al., 2004). All specimens were examined by light microscopy Axiovert 200M (Carl Zeiss, Jena, Germany). For automated morphometry analysis, the percentage of positive cells (brown staining) was determined with a computerized image analyzer KS-300 3.0 (Carl Zeiss, Jena, Germany). This equipment automatically detects positive cells determining their percentage per field. Five random fields per kidney were studied at 100 magnification (total area 1,584,000 square microns) comparing the different groups. Results were expressed as percentage (Cruz et al., 2007).

2.5. Statistical analyses Data were expressed as mean ± SEM. Data were analyzed with the software Prism 3.02 (GraphPad, San Diego, CA, USA) by one-way analysis of variance followed by Bonferroni multiple comparisons method. Kruskall–Wallis test followed by Dunn multiple comparisons method was used to compare the histological damage. p < 0.05 was considered significant.

3. Results We first investigated whether the antioxidant NDGA reduces or prevents renal dysfunction and structural injury induced by K2Cr2O7 administration. As shown in Figs. 1 and 2, after 48 h of a single K2Cr2O7 injection, the rats presented a marked reduction of renal function compared to

2.4. Histopathological and immunohistochemical analyses For light microscopy, kidney tissue was fixed by immersion in buffered formalin (pH 7.4) and embedded in paraffin. For histological analysis, sections (3 lm) were stained with hematoxylin and eosin (H&E). The histological profile of proximal tubules from 5 randomly selected fields (5 rats per experimental group) was recorded using KS-300 software (Carl Zeiss, Jena, Germany). The percentage of tubular area with histopathological alterations like swelling, cytoplasmic vacuolization, desquamation or necrosis was obtained. For immunohistochemistry, kidney sections (3 lm) were deparaffined and then boiled in Declere to unmask antigen sites; the endogenous activity of peroxidase was quenched with 0.03% H2O2 in absolute methanol. Kidney sections were incubated overnight at 4 °C with 1:200 dilution of anti 4-HNE and 1:70 dilution of anti 3-NT antibodies in phosphate buffered saline (PBS). Following removal of the primary antibodies and repetitive rinsing with PBS, slides were incubated with a 1:500 dilution of biotinylated goat anti-IgG secondary antibody. Bound antibodies were detected with avidin biotinylated peroxidase complex ABC-kit Vectastain and diaminobenzidine substrate. After appropriate washing in PBS, slides were counterstained with hematoxylin. All sections were incubated under the same conditions with the same concentration of antibodies and in the same running, so the immunostaining was comparable among the different experimental groups. For the

Fig. 1. Urinary excretion of (A) total protein (n = 8–11) and (B) N-acetylb-D-glucosaminidase (n = 4–8) in the groups of rats studied: (1) CT, (2) K2Cr2O7, (3) K2Cr2O7 + NDGA, and (4) K2Cr2O7 + DMSO. Rats were studied 2 days after K2Cr2O7 injection (15 mg/Kg). NDGA (17 mg/Kg/ day) or DMSO (33 mg/Kg/day) was administered by mini-osmotic pumps 1 day before and 2 days after K2Cr2O7 injection. Data are mean ± SEM. a p < 0.001 vs. CT, bp < 0.05 vs. K2Cr2O7.

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Fig. 2. (A) Serum creatinine (n = 6–10) and (B) activity of serum glutathione peroxidase (n = 10–16) in the groups of rats studied: (1) CT, (2) K2Cr2O7, (3) K2Cr2O7 + NDGA, and (4) K2Cr2O7 + DMSO. Rats were studied 2 days after K2Cr2O7 injection (15 mg/Kg). NDGA (17 mg/ Kg/day) or DMSO (33 mg/Kg/day) was administered by mini-osmotic pumps 1 day before and 2 days after K2Cr2O7 injection. Data are mean ± SEM. ap < 0.001, cp < 0.01 vs. CT, bp < 0.001 vs. K2Cr2O7.

CT group that was characterized by a significant increase in urinary excretion of total protein (P < 0.001) (Fig. 1A) and NAG (P < 0.001) (Fig. 1B) and in serum creatinine concentration (P < 0.001) (Fig. 2A) and by a significative decrease in serum GPx activity (P < 0.001) (Fig. 2B). NDGA administration attenuated significantly the increase in urinary excretion of total protein (P < 0.05) (Fig. 1A) and NAG (P < 0.05) (Fig. 1B) and in serum creatinine concentration (P < 0.001) (Fig. 2A) and the decrease in serum GPx activity (P < 0.001) (Fig. 2B). In contrast, the administration of DMSO, the vehicle in which NDGA was dissolved, was unable to modify the K2Cr2O7-induced changes in urinary excretion of total protein (Fig. 1A) and NAG (Fig. 1B), in serum creatinine concentration (Fig. 2A) and in serum GPx activity (Fig. 2B) (P > 0.05 in all cases). These data suggest that NDGA has a renoprotective effect in this experimental model, thus it was decided to further investigate whether this antioxidant confers histological protection. It was found that NDGA was also able to ameliorate significantly the area with histological damage in K2Cr2O7-treated rats (13.8 ± 1.2% in K2Cr2O7 + NDGA group vs. 56.2 ± 2.8% in K2Cr2O7 group and 52.7 ± 2.7% in K2Cr2O7 + DMSO group, n = 5, P < 0.01) (Fig. 3). Slices from CT group showed normal architecture. Slices from K2Cr2O7 and K2Cr2O7 + DMSO treated rats showed extensive tubular damage showing most of cortical tubules with epithelial atrophy and casts. Slices from K2Cr2O7 + NDGA treated rats had lesser tissue damage with few epithelial tubular cells affected (Fig. 3). Glomeruli from all groups remained

Fig. 3. Structural analysis from kidney sections (3 lm) from all studied groups. Rats were studied 2 days after K2Cr2O7 injection (15 mg/Kg). NDGA (17 mg/Kg/day) or DMSO (33 mg/Kg/day) was administered by mini-osmotic pumps 1 day before and 2 days after K2Cr2O7 injection. H&E, 400.

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unchanged as it was shown previously by electron microscopy (Pedraza-Chaverri et al., 1995). Thus, these findings clearly show the antioxidant NDGA ameliorates the

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K2Cr2O7-induced nephropathy. Taking into account the previous data and the scavenging properties of NDGA, it was decided to analyze whether the renoprotective effect

Fig. 4. Immunostaining for 4-HNE from kidney sections (3 lm) from all studied groups. Slices from K2Cr2O7 and K2Cr2O7 + DMSO treated showed positive 4-HNE immunostaining. In contrast, slices from K2Cr2O7 + NDGA treated rats showed less immunostaining. 100.

Fig. 5. Immunostaining for 3-NT from kidney sections (3 lm) from all studied groups. Slices from K2Cr2O7 and K2Cr2O7 + DMSO treated showed positive 3-NT immunostaining. In contrast, slices from K2Cr2O7 + NDGA treated rats showed less immunostaining. 100.

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Table 1 Quantitative data of the immunohistochemistry studies (%) K2Cr2O7

CT 4-HNE 3-NT

0.88 ± 0.08 3.54 ± 0.34

K2Cr2O7 + NDGA a

9.63 ± 0.39 17.42 ± 0.79a

b

1.29 ± 0.39 3.04 ± 0.33b

K2Cr2O7 + DMSO 9.75 ± 0.94a 16.18 ± 1.56a

Data are mean ± SEM, n = 4. a p < 0.001 vs. CT. b p < 0.001 vs. K2Cr2O7.

of NDGA was related with reduction of oxidative and nitrosative stress in K2Cr2O7-treated rats which were evaluated by renal immunohistochemistry for 4-HNE and 3NT, respectively. As shown in Figs. 4 and 5, a negative immunostaining for 4-HNE and 3-NT was observed in cortex sections of CT group. In contrast, a strong immunostaining for 3-NT and 4-HNE was observed in renal cortex from K2Cr2O7-treated rats (Figs. 4 and 5). NDGA administration protected the kidney of nitrosative and oxidative stress that was evinced by a weak immunoreactivity of 3-NT and 4-HNE in renal cortex from K2Cr2O7 + NDGA group (Figs. 4 and 5) which was confirmed by quantitative data (Table 1). The immunostaining for 4-HNE and 3-NT in the K2Cr2O7 + DMSO group was similar than that in the K2Cr2O7 group. 4. Discussion NDGA is a lignan present in high concentrations in the leaves of L. tridentate which has been used in the traditional medicine for the treatment of several illnesses (Arteaga et al., 2005, Table 2). NDGA is a remarkable antioxidant as has been shown in vivo (Anjaneyulu and Chopra, 2004; Ansar et al., 1999) and in vitro (FlorianoSanchez et al., 2006) studies. This work was designed taking into account the antioxidant ability of NDGA and the oxidative stress involved in K2Cr2O7-induced nephrotoxicity. The data obtained in the present work clearly show that the amelioration of K2Cr2O7-induced renal injury and oxidative stress by NDGA was statistically significant. NDGA was able to ameliorate not only the functional but Table 2 Use of Larrea tridentata in several diseases Disease

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also the structural renal damage induced by K2Cr2O7. The K2Cr2O7-induced decrease in serum GPx activity and increase in serum creatinine concentration and in the urinary excretion of total protein and NAG were ameliorated by NDGA. The K2Cr2O7-induced renal damage was not attenuated by DMSO, the vehicle of NDGA. Interestingly, higher doses of DMSO (4 g/Kg/12 h) have been used also to ameliorate renal damage in passive Heymann nephritis (Lotan et al., 1984) and in gentamicin-induced nephrotoxicity (Walker and Shah, 1988). The lack of DMSO effect may be explained by the low dose administered (33 mg/ Kg/day) compared to that used by Lotan et al. (1984) and Walker and Shah (1988). The data are relevant taking into account that occupational exposure to chromium has been associated with acute renal failure (Sharma and Singhal, 1978; Picaud et al., 1991; Franchini et al., 1978). This beneficial action of NDGA observed in the present study is consistent with its renoprotective effect described in rats with diabetic nephropathy (5 and 10 mg/Kg/day/4 weeks, s.c., Anjaneyulu and Chopra, 2004) and ferric nitrilotriacetate-induced nephrotoxicity in mice (40–100 mg/Kg/day/ 1 week by gavage, Ansar et al., 1999). In both cases the protective effect of NDGA was clearly associated with the amelioration of oxidative stress (Ansar et al., 1999; Anjaneyulu and Chopra, 2004) strongly suggesting that the antioxidant properties of NDGA are involved in its renoprotective effect. All four reducing equivalents from the two catechol groups in NDGA may be involved in their renoprotective and antioxidant properties described in this paper. It has been suggested that hydrogen atoms of the four phenolic hydroxyl groups react with ROS (Shahidi and Wanasundara, 1992). In addition, Abou-Gazar et al. (2004) suggested that the number and position of the hydroxy and methoxy substituents of the phenyl moieties may contribute to the antioxidant effect in an epoxylignan. The inhibition of cytotoxicity induced by t-butylhydroperoxide (Nakayama et al., 1991) and H2O2 (Nakayama et al., 1992) in mammalian cells is also consistent with the antioxidant effect of NDGA. Interestingly, the oxidative (4-HNE immunostaining) and nitrosative stress (3-NT immunostaining) observed in K2Cr2O7-treated rats was prevented by NDGA treatment. K2Cr2O7-induced renal injury has been associated with enhanced 3-NT immunostaining suggesting that ONOO, a strong oxidant and nitrating agent, is involved in the renal damage (Barrera et al., 2003; Pedraza-Chaverri et al., 2005). This observation was confirmed in the present study. The ability of NDGA to ameliorate 3-

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NT immunostaining may be related to their O and 2 ONOO scavenging properties (Floriano-Sanchez et al., 2006). It remains to be established if NDGA treatment is able to enhance non-enzymatic and enzymatic antioxidant system in the cells. 3-NT is thought to be a relatively specific marker of nitrosative damage mediated by ONOO,  which is produced by the reaction between O 2 and NO . The increase in 3-NT production may be secondary to  the increase of either O 2 or NO and it has been documented that K2Cr2O7 enhances O 2 production (Liu and Shi, 2001; Pritchard et al., 2000; Sugiyama, 1992). Taking into account the renoprotective effect of NDGA and the scavenging ability of this antioxidant for several ROS (Floriano-Sanchez et al., 2006), we are tempting to speculate that this antioxidant also may protect DNA from oxidative damage. The role of oxidative damage in K2Cr2O7-induced renal injury has been supported with the use of another antioxidants including: ascorbic acid, vitamin E, N-acetyl cysteine, and GSH (Sugiyama, 1992; Na et al., 1992; Appenroth and Winnefeld, 1998; Hojo and Satomi, 1991). In addition, the inhibition of GSH biosynthesis enhances the K2Cr2O7-induced renal injury (Sugiyama, 1992; Hojo and Satomi, 1991). In contrast with the beneficial effects described above for NDGA, adverse effects for this antioxidant have also been observed. In fact, NDGA has been banned in some countries as a food additive to preserve fats and butter by its side effects such as cystic nephropathy in rats (Evan and Gardner, 1979; Gardner et al., 1987; Goodman et al., 1970). Goodman et al. (1970) gave a diet containing 2% NDGA by 15 days and Gardner et al. (1987) gave a diet containing 2% NDGA by 22 days in normal rats. The approximate dose of NDGA for these animals was 1.1 g/Kg/day. Taking into account the above data, it is possible that the differences between the protective and deleterious effects of NDGA in vivo may be related to the daily dose given. The protective effect of NDGA in vivo has been observed with 5– 100 mg/Kg/day whereas the toxic effects have been observed with doses higher than 1 g/Kg/day. In conclusion, the data show that the ability of NDGA to ameliorate K2Cr2O7-induced renal injury is associated with its antioxidant properties. Acknowledgement This work was supported by CONACYT (Grant No. 48812). Axiovert 200 M confocal microscope was donated by Fundacion Gonzalo Rio Arronte IAP Me´xico. References Abou-Gazar, H., Bedir, E., Takamatsu, S., Ferreira, D., Khan, I.A., 2004. Antioxidant lignans from Larrea tridentata. Phytochemistry 65, 2499– 2505. Aiyar, J., Berkovits, H.J., Floyd, R.A., Wetterhahn, K.E., 1991. Reaction of chromium(VI) with glutathione or with hydrogen peroxide: identification of reactive intermediates and their role in chromium(VI)induced DNA damage. Environ. Health Perspect. 92, 53–62.

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