Preventive effect of tert-butylhydroquinone on cisplatin-induced nephrotoxicity in rats

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Preventive effect of tert-butylhydroquinone on cisplatin-induced nephrotoxicity in rats
  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:  Author's personal copy Preventive effect of   tert  -butylhydroquinone on cisplatin-induced nephrotoxicityin rats  Jazmin M. Pérez-Rojas a,b, ⇑ , Carlos Enrique Guerrero-Beltrán a , Cristino Cruz c , Dolores J. Sánchez-González d ,Claudia M. Martínez-Martínez d , José Pedraza-Chaverri a, ⇑ a Facultad de Química, Departamento de Biología, Universidad Nacional Autónoma de México, Mexico City, Mexico b Subdirección de Investigación Básica, Instituto Nacional de Cancerología (INCan), Mexico City, Mexico c Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico d Departamento de Biología Celular, Escuela Médico Militar, Universidad del Ejército y Fuerza Aérea, Mexico City, Mexico a r t i c l e i n f o  Article history: Received 15 February 2010Accepted 7 July 2011Available online 23 July 2011 Keywords:tert  -ButylhydroquinoneCisplatinOxidative stressKidney damage a b s t r a c t Cis-diamminedichloroplatinum II (CDDP)-induced nephrotoxicity is associated with the overproductionof reactive oxygen species.  tert  -Butylhydroquinone (tBHQ) is a compound widely used as food antioxi-dant. The purpose of this study was to investigate the ability of tBHQ to prevent the nephrotoxic effectofCDDPinratsaswellasthemechanismsinvolved.Thirty-sixWistarratsdividedinthefollowinggroupswere used: control, tBHQ (12.5mg/kg), CDDP (7.5mg/kg) and tBHQ+CDDP. Twenty-four h urine wascollected at the beginning and at the end of the experiment and the rats were sacrificed 72h afterCDDP-administration. Histological studies were performed and markers of renal function and oxida-tive/nitrosative stress were measured. In addition, the activity of the following antioxidant enzymeswas measured: glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione reductase (GR)and glutathione-S-transferase (GST). CDDP-induced renal dysfunction, structural damage and oxida-tive/nitrosative were prevented by tBHQ. In addition, tBHQ completely prevented the CDDP-induced fallin GPx and GST activities. In conclusion, the present study indicates that the antioxidant activity of tBHQ is associated with its nephroprotective effect against CDDP-induced acute kidney injury in rats.   2011 Elsevier Ltd. All rights reserved. 1. Introduction Cis-diamminedichloroplatinum II (CDDP) is an important che-motherapeutic agent that has beenwidelyusedfor itspotent cyto-toxic effects upon a variety of tumor types including testicular,ovarian, and cervical carcinoma (Fontanelli et al., 1992; Loehrerand Einhorn, 1984; Ozols, 1995; Panici et al., 1991). However,the administration of CDDP is associated with serious side effectsincluding nephrotoxicity and neurotoxic alterations (Gandaraet al., 1991). CDDP causes tubular injury, mainly at proximal tubu-lar level, through multiple mechanisms including hypoxia, oxida-tive stress, inflammation and apoptosis (Chirino et al., 2008a;Francescato et al., 2007; Yao et al., 2007). Despite its side effects,CDDP remains the drug of preference in chemotherapy, mainlybecauseofitsefficacyandlowcost.Thatiswhyitisveryimportantto counteract CDDP side effects to improve quality of life of pa-tients who are under this treatment regimen.OxidativestressisoneofthemostimportantfactorsinvolvedinCDDP-induced toxicity. Some researchers have been used naturalextracts, isolated compounds and new drugs to block oxidativestress by several ways with the purpose to prevent CDDP-inducedrenal damage (Ali and Al Moundhri, 2006; Chirino and Pedraza-Chaverri, 2009). In this context, it is important to employcompounds which are used for human beings, as is the case of  tert  -Butylhydroquinone (tBHQ), because they will be easily addedin therapy and also will be cheaper for the patient.tBHQ is a synthetic phenolic antioxidant (Kim et al., 2009) thatwasapprovedforhumanusebybothFoodandAgricultureOrgani-zation and World Health Organization (1999). Several groups haveused this compound because of its antioxidant properties. FavreauandPickett(1991)andRushmoreandPickett(1990)demonstrated that thetreatmentwithtBHQinducedmanygenes associatedwithresistance against oxidative stress and prevented hydrogen perox-ide (H 2 O 2 )-induced apoptosis in neuroblastoma IMR32 cells. Also,Hara and colleagues (2003) reported that tBHQ has a neuroprotec-tive effect against 6-hydroxydopamine induced oxidative stress inthe human neuroblastoma cell line SH-SY5Y. However, it is 0278-6915/$ - see front matter   2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.fct.2011.07.008 ⇑ Corresponding authors. Address: Laboratorio de Farmacología, Subdirección deInvestigación Básica, Instituto Nacional de Cancerología (INCan), Av. San Fernando22, Tlalpan 14000, Apartado Postal 22026, Mexico City, Mexico. Tel.: +52 55 56280400x303 (J.M. Pérez-Rojas). Facultad de Química, Edificio F, Universidad NacionalAutónoma de México, Mexico City, México. Tel./fax: +52 55 5622 3878(J. Pedraza-Chaverri). E-mail addresses: (J.M. Pérez-Rojas), (J. Pedraza-Chaverri).Food and Chemical Toxicology 49 (2011) 2631–2637 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage:  Author's personal copy unknown if tBHQ may protect the kidney from the CDDP-inducedrenal damage.For the above reasons, in the present study, we investigatedwhether the antioxidant properties of tBHQ may attenuate acuterenal failure, oxidative stress, and reduction in antioxidant en-zymes activities induced by CDDP in rats. 2. Methods  2.1. Reagents The Amplex Red  H 2 O 2 /peroxidase assay kit used to measureurinary excretion of H 2 O 2 , CDDP (Cat. No. P-4394) and tBHQ (Cat.No. 112941) were purchased from Sigma–Aldrich (St. Louis, MO,USA). Mayer’s Hematoxylin (Lillie’s Modification) (Cat. No. S3309)was from DAKO Corporation (Carpinteria, CA, USA). All otherchemicals used were of the highest quality available and obtainedfrom commercial sources.  2.2. Experimental design Thirty-six male Wistar rats were obtained from Harlan (MexicoCity) with a body weight of 286 ± 19 (mean ± SD), and were ran-domlydivided into four groups: (1) control (C) ( n  = 9), rats received  Table 1 Activity of CAT, SOD and GR in renal cortex. CAT (k/mgprotein)SOD (U/mgprotein)GR (U/mgprotein)Ct 0.253 ± 0.008 31.11 ± 3.4 0.082 ± 0.012tBHQ 0.214 ± 0.024 28.90 ± 2.1 0.081 ± 0.007CDDP 0.178 ± 0.018 a 31.66 ± 1.5 0.086 ± 0.004tBHQ + CDDP 0.109 ± 0.016 a,b 34.70 ± 1.9 0.077 ± 0.004CAT, catalase; SOD, superoxide dismutase; GR, glutathione reductase; Ct, control;tBHQ, tert-butylhydroquinone; CDDP, cisplatin. Values are mean ± SEM of 6–8 rats/group. a P   < 0.05  vs.  CT. b P   < 0.05  vs.  CDDP. Fig. 1.  Protective effect of tBHQ against CDDP-induced renal dysfunction in rats. (A)Serum creatinine, (B) blood urea nitrogen (BUN) and (C) creatinine clearance incontrol; tBHQ,  tert  -butylhydroquinone; CDDP, cisplatin and tBHQ + CDDP groups.Values are means ± SEM.  n  = 4–7.  ª P   < 0.001  vs.  Control;  b P   < 0.05  vs.  CDDP. Fig. 2.  Protective effect of tBHQ against CDDP-induced tubular injury in rats. (A)Urinary volume, (B) urinary excretion of N-acetyl- b - D -glucosaminidase (NAG) and(C) total protein excretion in control; tBHQ,  tert  -butylhydroquinone; CDDP,cisplatin and tBHQ + CDDP groups. Values are means ± SEM.  n  = 5–8.  ª P   < 0.05  vs. Control;  b P   < 0.05  vs.  CDDP.2632  J.M. Pérez-Rojas et al./Food and Chemical Toxicology 49 (2011) 2631–2637   Author's personal copy a single intraperitoneal injection of vehicle (saline solution). (2)tBHQ ( n  = 9), one day before vehicle injection, rats received tBHQ (12.5 mg/kg, i.p.) divided into three identical doses each 8 h. (3)CDDP ( n  = 9), rats were injected with a single intraperitoneal injec-tion of CDDP (7.5 mg/kg), which was previously dissolved in salinesolution (1.5 mg/mL). (4) tBHQ + CDDP ( n  = 9), one day beforeCDDP injection (7.5 mg/kg, i.p.), rats received tBHQ (12.5 mg/kg,i.p.) divided into three identical doses each 8 h. Before startingthe experiment and before the sacrifice, the rats were placed intometabolic cages to collect 24-h urine. In addition blood sampleswere obtained at the end of the experiment.Rats were placed in rooms at 22   C with 12:12-h light–darkcycle and free access to water. Three days after CDDP administra-tion rats were sacrificed by decapitation and blood was collected.A midline laparotomy was made; the right kidney was excised, fro-zen in liquid nitrogen, and kept at  80   C until the determinationswere performed. The left kidney was placed in 10% formalin bufferfor histopathological evaluation. All procedures followed were inaccordance with our institutional guidelines.The dose of tBHQ was chosen after the performance of previ-ous experiments using the following doses: 12.5 mg/kg one daybefore CDDP administration, 12.5 mg/kg for 5 days prior of CDDPadministration and 25 mg/kg body weight 5 days prior to theinjection of CDDP. The following parameters were evaluated:serum creatinine, blood urea nitrogen (BUN), creatinine clear-ance (CCr), and urinary excretion of total protein and N-acetyl- b -D-glucosaminidase (NAG). tBHQ (12.5 mg/kg) given one dayprevious to drug administration, showed the highest renoprotec-tive effect against CDDP-induced nephrotoxicity (data not show).Based on the above data, that dose was chosen to perform thepresent study.  2.3. Analytical methods Renal function was evaluated by measuring BUN, serum creati-nine, creatinine clearance and urinary excretion of total proteinand NAG by methods previously described (Chirino et al., 2008a). Fig. 3.  Protective effect of tBHQ against CDDP-induced renal histological alteration in rats. (A) Histological profiles were detected by H&E staining in rats treated with salinesolution (Control); tBHQ,  tert  -butylhydroquinone; CDDP, cisplatin and tBHQ + CDDP. CDDP induced necrosis(asterisks), vacuolization (arrowheads) and tubular casts (arrow).(B) Histological damage expressed as percentage of tubular area. x400 magnification. Values are means ± SEM.  n  = 4.  ª P   < 0.0002  vs.  CDDP.  J.M. Pérez-Rojas et al./Food and Chemical Toxicology 49 (2011) 2631–2637   2633  Author's personal copy  2.4. Histological studies Kidney sections were fixed in 10% neutral buffered formalinsolution and embedded in paraffin (Pérez-Rojas et al., 2009). Sec-tions of 3 l m of thickness were obtained and stained with hema-toxiline-eosin (H&E). A quantitative histological damage wasdetermined by using a Leica Qwin Image Analyzer (Cambridge,England). The histological profiles of thirty proximal tubules ran-domly selected per rat were recorded. The percentage of tubuleswith histopathological alterations like swelling, cytoplasmic vacu-olization, desquamation, or necrosis was obtained. Total surfacearea of tubular cells in square microns was determined, the surfacearea occupied by vacuoles was measured, and the percentage of this affected area was calculated.  2.5. Oxidative stress markers Urinary excretion of H 2 O 2  was measured with the Amplex Red  method (Pérez-Rojas et al., 2009). Malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were measured in the renal cortex usinga standard curve of trimethoxypropane (Gerard-Monnier et al.,1998). The content of protein carbonyl groups, an index of oxidizedproteins, was measured using 2,4-dinitrophenylhydrazine (Maldo-nado et al., 2003).  2.6. Immunohistochemical studies Immunohistochemical studies of 4-HNE, a marker of lipid per-oxidation, and of 3-nitro- L  -tyrosine (3-NT), a marker of nitrosativestress, were performed as previously described (Chirino et al.,2008a; Pérez-Rojas et al., 2009).  2.7. Activity of antioxidant enzymes Glutathione peroxidase (GPx) activity was measured by the dis-appearance of NADPH at 340 nm in a coupled assay containingH 2 O 2 , GSH and glutathione reductase (GR) (Maldonado et al.,2003). GR activity was measured by the disappearance of NADPHat 340 nm in a reaction mixture containing oxidized glutathioneas a substrate (Maldonado et al., 2003). Total superoxidedismutase(SOD) was measured at 560 nm using the superoxide anion gener-ator system xanthine/xhanthine oxidase and nitroblue tetrazoliumas the indicator molecule (Maldonado et al., 2003). Catalase (CAT)activity was assayed at 240 nm by a method based on the disap-pearance of H 2 O 2  (Maldonado et al., 2003). Glutathione-S-transfer-ase (GST) activity was assayed at 340 nm in a mixture containingglutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as pre-viously described (Chirino et al., 2008a).  2.8. Statistical analysis Results were expressed as mean ± SE. Data were analyzed byone-way ANOVA followed by student Newman-Keuls test. Non-paired  t  -test was used to compare the quantitative histologicaldamage. GraphPad Prism 2.01 software (San Diego, CA, USA) wasused. A  p -value < 0.05 was considered statistically significant. Fig. 4.  Protective effect of tBHQ against CDDP-induced increase in renal immunostaining of 3-NT and 4-HNE in rats. (A) Representative microphotographs of 3-NT. (B)Percentage area of 3-NT abundance. (C) Representative microphotographs of 4-HNE. (D) Percentage area of 4-HNE abundance. tBHQ,  tert  -butylhydroquinone; CDDP, cisplatin.Values are mean ± SEM.  n  = 4.  ª P   < 0.001  vs.  Ct;  b P   < 0.01  vs.  CDDP.2634  J.M. Pérez-Rojas et al./Food and Chemical Toxicology 49 (2011) 2631–2637 
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