Effect of diabetes on biodistribution, nephrotoxicity and antitumor activity of cisplatin in mice.

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Effect of diabetes on biodistribution, nephrotoxicity and antitumor activity of cisplatin in mice Marcia C. da Silva Faria a, Neife A.G. dos Santos a, Maria A. Carvalho Rodrigues a, Jairo Lisboa Rodrigues b, Fernando Barbosa Junior a, Antonio Cardozo dos Santos a,⇑ a b

Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto – USP, 14040-903 Ribeirão Preto, SP, Brazil Instituto de Ciência e Tecnologia do Mucuri, Universidade Federal dos Vales do Jequitinhonha e Mucuri, Teófilo Otoni, MG, Brazil

a r t i c l e

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Article history: Received 19 September 2014 Received in revised form 20 December 2014 Accepted 7 January 2015 Available online xxxx Keywords: Cisplatin Diabetes Sarcoma 180 Nephrotoxicity Antitumor activity Biodistribution

a b s t r a c t Both types of diabetes are associated with higher incidence of some types of cancer. Treating cancer in diabetic patients without aggravating diabetes-related complications is a challenge for clinicians. Additionally, little is known about how diabetes affects the treatment of cancer. One of the most effective chemotherapeutic drugs is cisplatin, which is nephrotoxic. Studies suggest that diabetes acts as a protective factor against the nephrotoxicity of cisplatin, but the mechanisms involved have not been elucidated yet. This renal protection has been attributed to decreased accumulation of cisplatin in the kidneys, which could be associated with deficient active transport of proximal tubular cells or to pharmacokinetic alterations caused by diabetes. However, it is uncertain if diabetes also compromises the antitumor activity of cisplatin. To address this issue, we developed a mouse model bearing cisplatin-induced nephrotoxicity, Sarcoma 180 and streptozotocin-induced diabetes. Four groups of treatment were defined: (i) control, (ii) diabetic, (iii) cisplatin and (iv) diabetic treated with cisplatin. The following parameters were evaluated: renal function, oxidative stress, apoptosis, renal histopathology, tumor remission, survival rate, genotoxicity and platinum concentration in tumor and several organs. Results indicate that diabetes protects against the renal damage induced by cisplatin, while also compromises its antitumor effectiveness. This is the first study to demonstrate this effect. Ó 2015 Elsevier Ireland Ltd. All rights reserved.

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1. Introduction

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Both types of diabetes mellitus (DM) have been associated with increased risk of cancer. Type 2 diabetes is mainly associated with increased risk of breast, colon, pancreatic and liver cancer, while type 1 diabetes is associated with increase in cervical, stomach, pancreatic, and endometrial cancer. The increased risk of cancer in diabetes has been associated mainly with hyperglycemia and hyperinsulinemia [50]. Hyperglycemia supplies the high demand for glucose of tumor cells [78], while insulin is a hormonal stimulator for cellular proliferation [48,80]. In addition, there is a clear relationship between blood glucose reduction and remission of malignant tumors [40]. A study in a model of breast cancer in mice showed that higher values of glycaemia were associated with higher mortality of those animals [58]. It is known that the kidneys

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⇑ Corresponding author at: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, USP, Avenida do Café s/n, 14040-903 Ribeirão Preto, SP, Brazil. Tel.: +55 1636024159; fax: +55 1636024725. E-mail address: [email protected] (A.C. dos Santos).

of diabetic rats are protected against the nephrotoxicity induced by structurally different compounds, including cisplatin; however, the events underlying this protection are not clear [74,66,67,18]. Cisplatin (cis-diamminedichloroplatinum II) is one of the most effective chemotherapeutic agents, but the exploitation of its full therapeutic potential is limited predominantly due to its nephrotoxicity [16,21,38,42]. The kidney is the main organ of excretion of cisplatin and it is also the major site of cisplatin accumulation [64]. The concentration of cisplatin in proximal tubular epithelial cells is five times higher than in the serum [41]. The resistance to nephrotoxicity is proportional to the duration and severity of the diabetic state; low resistance is observed in rats after a week of experimentally induced diabetes, and total resistance is found after six weeks of uncontrolled diabetes experimentally induced by streptozotocin. The resistance against the nephrotoxicity induced by cisplatin has been found in both insulin-dependent and insulin-independent diabetes [66,10,11]. The treatment of diabetic rats with insulin for 21 days abolishes the protection against cisplatin-induced nephrotoxicity and increases the susceptibility of animals to this toxicity [63]. The attenuation of cisplatininduced nephrotoxicity in diabetic rats has been associated with

http://dx.doi.org/10.1016/j.cbi.2015.01.027 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: M.C. da Silva Faria et al., Effect of diabetes on biodistribution, nephrotoxicity and antitumor activity of cisplatin in mice, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.01.027

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decreased platinum concentration in plasma [49]. It has also been associated with decreased accumulation of platinum in the kidneys, which has been attributed to deficiencies in the active transport of the drug in tubular epithelial cells [10,62]. However, it is uncertain whether the mechanisms involved in the diabetic renal protection interfere with the antitumor activity of cisplatin and this has been addressed in the present study. For this purpose, we developed an experimental mouse model bearing cisplatininduced nephrotoxicity, Sarcoma-180-tumor and streptozotocininduced diabetes, which, to our knowledge, has not been previously studied.

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2. Materials and methods

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2.1. Chemicals

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High purity reagents were used (analytical grade minimum). Reagents were obtained from Sigma–AldrichÒ, unless specified differently. Type I ultra-pure water was obtained in the purification system by reverse osmosis (Rios DI-3), followed by purification in Milli-Q Gradient system (Millipore, Bedford, USA). Cisplatin solution (1 mg/ml) was prepared in saline (0.9% NaCl) and streptozotocin solution (15 mg/ml) was prepared in 50 mM sodium citrate buffer, pH 4.5. Both were prepared immediately before use.

After euthanasia, the kidneys were immediately removed, immersed in cold phosphate buffered saline (PBS) and kept in ice bath. The cortex was carefully separated from the medullae, weighed and homogenized in PBS (1:10) by using a Potter–Elvehjem type homogenizer (three 15-s cycles, 1-min intervals). The homogenate was centrifuged at 350g for 10 min (4 °C) and the supernatant was removed for protein determination and oxidative stress assays. Homogenate protein concentration was determined by a spectrophotometric (Biuret, 540 nm) method [12].

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2.2. Tumor cells (Sarcoma 180, S-180)

2.6. Reduced glutathione (GSH)

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The method previously described by Hissin and Hilf [31] was used. Reduced glutathione was determined in kidney homogenate by fluorimetry (excitation 350 nm, emission 420 nm). A calibration curve containing 7.5, 15, 30, 60, 150 and 300 mM of reduced glutathione was used to calculate the concentrations of GSH in the samples.

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2.7. Lipid peroxidation

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Tumor cells were cultured in the ascites of mice for 8 days prior to inoculation in other mice for development of solid tumor. To preserve the tumor cell line, cells were grown (at 37 °C in a humidified 5% CO2-containing atmosphere) in DMEM culture medium (Dulbeccos Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS) and 0.1% of antibiotic solution (PNS, 5 mg/ml of penicillin, 5 mg/ml neomycin and 10 mg/ml streptomycin). For preservation Sarcoma 180 cells were frozen in the same culture medium previously described supplemented with 20% FBS and 10% DMSO (dimethylsulfoxide) [72,22].

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2.3. Animal model

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Swiss male adult mice (25–30 g) were housed four per cage and were maintained in a 12-h light/dark cycle in a temperature (22– 24 °C) and humidity (45–55%) controlled facility. Standard chow and water were provided ad libitum. Animals were separated into 4 groups of treatment (n = 8 in each group) as follows:

TBARS (thiobarbituric-acid-reactive substances) were determined in kidney homogenate by using the method previously described by Buege and Aust [9]. The absorbance at 535 nm was monitored and calculations were performed based on a calibration curve containing 0.5, 1, 3, 10, 12.5, 15, 20 and 50 lM of 1,1,3,3-tetramethoxypropane (TMOP). 2.8. NADPH oxidation

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The method previously described by Lund et al. [46] was used. NADPH oxidation was monitored by fluorimetry (emission: 450 nm, excitation: 340 nm).

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2.9. Histopathology

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Kidneys were fixed in paraformaldehyde (4%), dehydrated through a series of graded ethanol baths (70%, 95% and 100%) and embedded in paraffin. Tissue sections (4 lm) were placed on slides, stained with H&E and the morphology was analyzed under light microscopy (200). The percentage of damaged renal tubules was calculated by counting ten different fields in the outer stripe of the outer medulla and adjacent cortex per slide. The analysis of longitudinal sections of renal tissue was performed by optical microscopy (400). The degree of renal damage was analyzed based on a scale from 0 to 4, defined as follows: 0 = normal; 0.5 = small focal areas of cellular infiltration and tubular injury; 1 = lesion involving less than 10% of the cortex; 2 = injury involving 25% of the cortex; 3 = injury involving 25–50% of the cortex; and 4 = extensive damage involving more than 75% of cortex [69].

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(a) Control (C): Sarcoma180-tumor bearing mice without treatment. (b) CIS: Sarcoma180-tumor bearing mice treated with cisplatin. (c) DB: Sarcoma180-tumor bearing diabetic mice without treatment. (d) DB + CIS: Sarcoma180-tumor-bearing, diabetic mice treated with cisplatin.

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Diabetes was induced by an injection (150 mg/kg, injection volume: 300 lL) of streptozotocin (15 mg/ml). After 10 days 80% of animals presented levels of blood glucose above 250 mg/dl and were considered diabetic. Sarcoma-180 cells from the ascites of mice were injected (100 lL) subcutaneously in the ventral region of other mice, just above the femoral muscle. A solid tumor developed after 8 days of inoculation [70]. Then, an intraperitoneal injection (600 lL) of cisplatin (1 mg/ml) was administered in a single dose (20 mg/kg). The survival rate assays were terminated 170 days after cisplatin treatment. In all other assays the animals were euthanized after 72 h of cisplatin treatment. Blood samples were collected for tests of renal function. The tumor and organs

(kidneys, brain, testes, liver, skeletal muscle, heart and lung) were removed immediately for the determination of platinum levels. Research protocols were in strict accordance with the ‘‘Ethical principles and guidelines for experiments on animals’’ of the Swiss Academy of Medical Sciences and Swiss Academy of Sciences, and were approved by the Ethics Committee on Animal Use (CEUA) of Ribeirão Preto Campus – USP.

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2.4. Assessment of the renal function

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Urea and creatinine levels were measured in plasma by using commercially available diagnostic kits (LabtestÒ, MG, Brazil).

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2.5. Preparation of kidney homogenate and protein determination

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2.10. Survival rate

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The groups of animals (n = 8) were monitored daily for death events until 170 days after cisplatin treatment, after which the experiment was terminated. The analysis was performed as previously described [25].

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2.11. Tumor mass

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After 72 h from the beginning of the treatment the animals were euthanized, tumors were removed and weighed.

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2.12. Genotoxicity of cisplatin

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In order to determine the interference of diabetes in the genotoxicity of cisplatin and consequently in its antitumor activity, we determined the frequency of micronuclei in peripheral blood and the nuclear division index (NDI) in the bone marrow, as described below.

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2.12.1. Frequency of micronuclei in peripheral blood This assay was performed according to the method previously described [30]. After 24 and 72 h of the administration of cisplatin blood was collected from the caudal vein and smeared on a microscope slide. After 24 h, it was fixed with methanol and dried for 10 min at room temperature. The slides were stained with acridine orange (125 lg/ml, Sigma–Aldrich) 1 min prior to analysis with a fluorescence microscope Olympus BX 51 (488 nm, blue light, yellow filter, 400). The presence of micronuclei in 2000 polychromatic erythrocytes (immature) was analyzed [13].

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2.12.2. Nuclear division index (NDI) in bone marrow The femur was removed and its ends were cut to give access to the marrow canal, which then was flushed with a syringe containing 2.0 ml of fetal bovine serum. The bone marrow cells were gently mixed with the serum by repeatedly pulling and pushing with the syringe [65,47]. The cells were centrifuged at 700g for 5 min and the supernatant was removed almost completely. The pellet was mixed with the small residual volume of the supernatant and smeared on a microscope slide. The material was fixed in absolute methanol for 5 min and stained with Giemsa diluted in phosphate buffer pH 6.8 (1:20) for 10 min. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were counted out of 500 erythrocytes and the following equation was applied: NDI = PCE/(PCE + NCE) [1].

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2.13. Concentration of platinum in tumor and organs

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A sample of tissue was weighed (15 a 85 mg), mixed with 1 ml of a 50% solution (v/v) of tetramethylammonium (TMAH) hydroxide and incubated for 48 h at room temperature. Next, 14 ml of diluent solution (0.5% HNO3 (v/v) and 0.01% (v/v) Triton X-100) were added. The 195 Pt isotope was monitored by mass spectrometry with inductively coupled plasma (ICP-MS ELAN DRCII, PerkinElmer) and quantification was performed based on a calibration curve (2; 5; 10:20 ppb platinum) prepared in the same diluent solution. The platinum concentration was expressed in mg/g tissue [5].

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2.14. Immunohistochemistry for apoptosis evaluation

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Kidney sections were dried overnight at 37 °C, de-waxed in xylene and re-hydrated in graded series of ethanol (100%, 95%, 70%), H2O and PBS. For antigen retrieval tissue sections were treated with proteinase K (20 lg/ml in 0.05 M Tris–HCl, 0.01 M CaCl2, pH 8.0 buffer, SigmaÒ) for 25 min at room temperature (RT).

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Endogenous peroxidase activity was blocked by immersion in 3% hydrogen peroxide in PBS (30 min, RT). Unspecific bindings were blocked with 2% horse serum in PBS (50 min, RT). Afterwards the following procedures were performed.

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2.14.1. Determination of caspase-3 and Bcl-2 Sections were incubated with primary antibodies (monoclonal anti-caspase-3 or anti-Bcl-2, Santa Cruz Biotechnology, 4 lg/ml in bovine serum albumin 1 mg/ml) for 12 h in a sealed humidity chamber. Immunoreactivity was detected by using peroxidase linked secondary antibody (poly – Max, Invitrogen). Visualization of antibody localization was enhanced by diaminobenzidine hydrochloride (DAB). Sections were rinsed three times with PBS for 5 min, counterstained with hematoxylin (40 s), dehydrated in ethanol, cleared in xylene, and mounted using Entelan (MerckÒ) mounting medium and glass coverslips. Sections were analyzed under light microscopy (400 magnification). Quantitation was performed by using ‘‘Image J’’ (1.44p Java 1.6, NIH) public access program.

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2.14.2. In situ direct DNA fragmentation (TUNEL) assay The DNA fragmentation was assessed in kidney sections (4 lm) by the TUNEL assay (ApopTagÒ, Peroxidase In Situ Apoptosis Detection Kit, ChemiconÒ), according to the manufacturer’s protocol. Cells that were positively labeled were quantified in ten different fields per slide under light microscopy (400 magnification). Positive labeled mammary gland cells provided in the kit were used as positive controls.

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2.15. Statistical analysis

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Data were expressed as mean ± SEM. Statistical analyses were conducted by the nonparametric Student’s t test (Mann–Whitney) for comparison between each pair of groups (CIS C; DB C and DB + CIS CIS), except for histology in which Wilcoxon matched pairs test was applied. For the survival assay, the Kaplan–Meier survival method was employed and the Logrank test was used for the comparison of the survival curves. All analyses were performed in the GraphPad Prism Software, version 6 for Windows (GraphPad Software, San Diego, CA, USA). Statistical significance was defined as p < 0.05.

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3. Results

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3.1. Inoculation of Sarcoma 180 cells

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Initially, the peritoneal cavity fluid containing S-180 tumor cells was diluted with PBS to obtain 5 107 cells/ml, and 100 ll of this solution were injected in recipient animals, but cell viability was low. In order to improve cell viability this procedure was modified as follows: the peritoneal fluid (average concentration: 2 108 cells/ml) was collected and directly injected into the recipient animals. This new procedure reduced from 15–20 days to 8 days the time necessary for tumor to reach an approximate size of 0.96 0.75 0.81 mm. Different regions of the body of mice were tested for cell inoculation by subcutaneous injection. The ventral region just above the femoral muscle was chosen [57,70] because in this region the tumor could be easily identified and removed, as shown in Fig. 1.

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3.2. Treatment with streptozotocin to induce diabetes

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Different doses of streptozotocin at different dose regimen were tested. Intraperitoneal injection of 80 mg/kg once a day for five consecutive days caused death before the end of experiment.

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Fig. 1. Subcutaneous Sarcoma S-180 developed (a) above the femoral muscle and (b) at the femoral muscle. In model (a) a well-defined tumor is formed. Experimental conditions are described in Section 2.

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Besides that, it has been reported that streptozotocin at multiple dose regimen might cause nephrotoxicity [75], which would add to the renal damage induced by cisplatin and jeopardize our experimental model. Therefore, a single dose of 150 mg/kg [32] was chosen. Animals whose blood glucose levels were at least 250 mg/dl at 10 days after streptozotocin injection were considered diabetic [36]. Approximately 15% of animals treated with this dose of streptozotocin were not diabetic, which has also been reported in another study [75].

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3.3. Treatment with cisplatin: induction of nephrotoxicity

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We evaluated different doses of cisplatin injected at the 8th day after the inoculation of S-180 cells. Initially 8 mg/kg [57,33] was tested; however, this concentration did not cause nephrotoxicity. Then, animals were treated with different doses of cisplatin (15, 20, 25, 30, 35 and 40 mg/kg) at the 8th day after the inoculation of S-180 cells. The dose 30 mg/kg caused nephrotoxicity, but it caused excessive mortality in the group DB + CIS. The dose 15 mg/kg was not able to cause nephrotoxicity, i.e., there was no significant difference in urea and plasma creatinine between the CIS and C groups. The lowest dose that caused nephrotoxicity was 20 mg/kg, which has also been used by other authors [44].

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3.4. Renal function

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BUN (Fig. 2A) and plasma creatinine (Fig. 2B) were significantly higher in CIS (474.25 ± 62.95 and 2.23 ± 0.42, respectively) than in

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C (51.90 ± 2.22 and 0.26 ± 0.01 mg/dl, respectively) which indicates the renal damage induced by cisplatin and validates the experimental model of nephrotoxicity. There was a significant decrease in the concentrations of BUN and creatinine in DB + CIS (101.60 ± 10.95 and 0.42 ± 0.03 mg/dl, respectively) as compared to CIS (474.25 ± 62.95 and 2.23 ± 0.42, respectively), which supports the renal protection conferred by diabetes that has been reported in other studies [74,66,67]. There was no significant difference in BUN and plasma creatinine between groups C (51.90 ± 2.22 and 0.26 ± 0.01 mg/dl, respectively) and DB (57.38 ± 4.75 and 0.26 ± 0.04 mg/dl, respectively).

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3.5. Lipid peroxidation

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Lipid peroxidation is a good marker of cellular dysfunction in oxidative stress (Hermes-Lima et al., 1995). After lipid peroxidation various compounds are formed by degradation of oxidized lipids. A classic and common method for quantifying these compounds is based on a colorimetric reaction with thiobarbituric acid [9]. In this study, the oxidation of membrane lipids was determined by assessing the thiobarbituric-acid-reactive substances (TBARS) in fresh samples. There was a significant increase in TBARS in CIS (0.79 ± 0.08 nmol/mg protein) as compared to C (0.12 ± 0.01 nmol/mg protein). TBARS decreased significantly in DB + CIS (0.06 ± 0.01 nmol/mg protein) as compared to CIS (0.79 ± 0.08 nmol/mg protein). There was no significant difference in TBARS between DB (0.17 ± 0.05 nmol/mg protein) and C (0.12 ± 0.01 nmol/mg protein). Results are presented in Fig. 3.

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3.6. GSH

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CIS (1.34 ± 0.18 nmol/mg protein) and DB (1.32 ± 0.17 nmol/mg protein) presented a significantly decreased concentration of GSH in relation to C (2.19 ± 0.21 nmol/mg protein). No significant difference was observed between DB + CIS (1.45 ± 0.10 nmol/mg protein) and CIS (1.34 ± 0.18 nmol/mg protein). Results are presented in Fig. 4.

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3.7. NADPH

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No significant differences were observed between the pairs of compared groups: CIS (91.94 ± 0.31) or DB (94.92 ± 0.25) versus C (100.0 ± 0.34); and DB + CIS (88.08 ± 0.17) versus CIS (91.94 ± 0.31). Results are presented in Fig. 5.

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Fig. 2. (A) Blood Urea Nitrogen (BUN). (B) Plasma creatinine. Values are expressed as mean ± SEM (n = 8) of assays performed in triplicates. C, control; CIS, cisplatin group; DB, diabetic group and DB + CIS, diabetic group treated with cisplatin. ⁄Significantly different from control (p < 0.05); #significantly different from CIS (p < 0.05). Experimental conditions are described in Section 2.


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Fig. 3. Lipid peroxidation in mice kidney homogenates. Values are expressed as mean ± SEM (n = 8) of assays performed in triplicates. C, control; CIS, cisplatin group; DB, diabetic group and DB + CIS, diabetic group treated with cisplatin. ⁄ Significantly different from control (p < 0.05); #significantly different from CIS (p < 0.05). Experimental conditions are described in Section 2.

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3.8. Histopathology of the renal tissue

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The histopathological changes of renal tissue caused by cisplatin were evaluated in hematoxylin-and-eosin stained (H&E) sections. The groups C (1.64 ± 0.32) and DB (2.12 ± 0.22) did not show significantly different scores of tubular damage, confirming that the dose regimen of streptozotocin did not induce renal injury. The CIS group (4.00 ± 0.05) showed a lesion score significantly higher than C (1.64 ± 0.32). These data are consistent with the renal function results, and confirm cisplatin-induced nephrotoxicity in our experimental model. DB + CIS (2.57 ± 0.20) showed a significantly reduced lesion score as compared to CIS (4.00 ± 0.05). The lesion scores are presented in Table 1 and the photomicrographs are presented in Fig. 6.

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3.9. Survival rate

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The survival curves of the groups were significantly different (Logrank test, P value < 0.0001). The median survival was signifi-

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Fig. 4. GSH in mice kidney homogenates. Values are expressed as mean ± SEM (n = 8) of assays performed in triplicates. C, control; CIS, cisplatin group; DB, diabetic group and DB + CIS, diabetic group treated with cisplatin. ⁄Significantly different from control (p < 0.05). Experimental conditions are described in Section 2.

Fig. 5. NADPH in mice kidney homogenates. Values are expressed as mean ± SEM (n = 8) of assays performed in triplicates. C, control; CIS, cisplatin group; DB, diabetic group and DB + CIS, diabetic group treated with cisplatin. No significant differences were observed between CIS versus C, DB versus C or DB + CIS versus CIS. The intensity of fluorescence was normalized to 100% in control. Experimental conditions are described in Section 2.

Table 1 Histopathology: lesion scores in the renal tissue. Groups

Lesion scores

C CIS DB + CIS DB

1.64 ± 0.32 4.00 ± 0.02* 2.57 ± 0.20# 2.12 ± 0.22

Values are expressed as mean ± SEM (n = 8). The longitudinal sections of kidney tissue were analyzed according to the degree of injury of the cortex by using a scale from 0 to 4. Scores meaning: 0 = normal; 0.5 = some focal areas of cellular infiltration and tubular injury; 1 = lesion involving less than 10% of the cortex; 2 = injury involving 25% of the cortex; 3 = injury involving 50–75% of the cortex; and 4 = extensive damage involving more than 75% of the cortex. * Significantly different from C (p < 0.05). # Significantly different from CIS (p < 0.05). No significant difference was observed between C and DB. Experimental conditions are described in Section 2. C, control; CIS, cisplatin group; DB, diabetic group; DB + CIS, diabetic group treated with cisplatin.

cantly lower in DB + CIS (17.5) than in CIS (48.5) probably due to the impaired antitumor action of cisplatin in the DB + CIS group. There was no significant difference between the median survival in the control group (65) and in the CIS group (62.5), however, while all the animals in the control group died within 100 days, 25% of animals in CIS survived at least 170 days, when the assay was terminated. Animals in the DB group died within 55 days and in the DB + CIS group died within 27 days, which indicates that cisplatin treatment was not beneficial in this group. The survival curves are depicted in Fig. 7.

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3.10. Tumor mass

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Tumor mass was significantly reduced in CIS (0.19 ± 0.02 g) as compared to C (0.61 ± 0.07 g), which demonstrates the antitumor efficacy of cisplatin. There was no significant difference in tumor mass between groups C (0.61 ± 0.07 g) and DB (0.69 ± 0.14 g), both without cisplatin treatment. The CIS + DB group showed significantly higher tumor mass (0.32 ± 0.02 g) as compared to CIS (0.19 ± 0.02). These data suggest that the antitumor activity of cisplatin was affected by diabetes (Fig. 8).

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Fig. 6. Histopathology. Representative photomicrographs of H&E stained longitudinal sections of kidney tissue (400). Groups: (A) control; (B) CIS; (C) DB and (D) DB + CIS. Experimental conditions are described in Section 2. CIS, cisplatin; DB, diabetic; DB + CIS, diabetic treated with cisplatin.

Table 2 Concentration of platinum in different tissues (lg/g).

Fig. 7. Survival rate. The total number of animals in each group (n = 8) was normalized to 100%. Experiment was terminated 170 days after cisplatin treatment and only in the CIS group there were animals alive. CIS, cisplatin; DB, diabetic; DB + CIS, diabetic treated with cisplatin. Experimental conditions are described in Section 2.

TISSUES

C

CIS

CIS + DB

DB

Tumor Kidneys Liver Brain Testicles Heart Muscle Lungs

6LOD 6LOD 6LOD 6LOD 6LOD 6LOD 6LOD 6LOD

17.09 ± 1.20* 32.72 ± 4.64* 325.81 ± 15.70* 0.668 ± 0.07* 8.76 ± 0.83* 5.44 ± 0.59* 4.86 ± 0.33* 13.16 ± 1.57*

7.98 ± 1.24# 12.25 ± 1.28# 21.98 ± 2.73# 0.389 ± 0.02# 5.71 ± 0.64# 2.7 ± 0.35# 2.65 ± 0.37# 6.89 ± 0.97#

6LOD 6LOD 6LOD 6LOD 6LOD 6LOD 6LOD 6LOD

C, control; CIS, cisplatin group; DB, diabetic group and DB + CIS, diabetic group treated with cisplatin; LOD, low limit of quantitation = 0.006 lg/g. * Significantly different from C (p < 0.05). # Significantly different from CIS (p < 0.05). No significant difference was observed between C and DB. Data are expressed as mean ± SEM (n = 8). Experimental conditions are described in Section 2.

Table 3 Nuclear division index (NDI) in bone marrow. Groups

NDI

C CIS DB + CIS DB

0.65 ± 0.03 0.14 ± 0.03* 0.51 ± 0.04# 0.68 ± 0.03

C, control; CIS, cisplatin group; DB, diabetic group and DB + CIS, diabetic group treated with cisplatin. * Significantly different from C (p < 0.05). # Significantly different from CIS (p < 0.05). No significant difference was observed between C and DB. Data are expressed as mean ± SEM (n = 8). Experimental conditions are described in Section 2.

Fig. 8. Tumor mass. Values are expressed as mean ± SEM (n = 8). C, control; CIS, cisplatin group; DB, diabetic group and DB + CIS, diabetic group treated with cisplatin. ⁄Significantly different from control (p < 0.05); #significantly different from CIS (p < 0.05). Experimental conditions are described in Section 2.

3.11. Platinum biodistribution

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The biodistribution of cisplatin in the tumors and in several organs was evaluated based on the concentration of platinum. In groups C and DB no platinum was detected (LOD = 0.006 lg/g). The concentrations of platinum in the tumor and in all the organs

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Fig. 9. Micronucleus assay. (A–D) Representative photomicrographs of peripheral blood and bone marrow cells of the CIS group. (A and C) peripheral blood cells (24 htreatment) stained with acridine-orange; (B) bone marrow cells (72 h-treatment) stained with Giemsa; (D) peripheral blood cells (72 h- treatment) stained with acridineorange. Magnification: (A) 1000; (B–D) 400. (E) Graphical representation of the frequency of polychromatic erythrocytes with micronuclei in peripheral blood after 24 h of cisplatin treatment. Values are expressed as mean ± SEM (n = 8) of MNPCE/1000 erythrocytes. Experimental conditions are described in Section 2. ⁄Significantly different from control (p < 0.05); #significantly different from CIS (p < 0.05) MNPCE, polychromatic erythrocytes with micronuclei; PCE, polychromatic erythrocyte; NCE, normochromatic erythrocyte; C, control; CIS, cisplatin group; DB, diabetic group and DB + CIS, diabetic group treated with cisplatin.

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were significantly higher in CIS than in C (p < 0.05) and significantly lower in CIS + DB than in CIS (Table 2).

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After 24 h of cisplatin administration, the CIS group presented increased number of micronuclei (MN) as compared to C. After 72 h of cisplatin administration, it was not possible to quantify the number of polychromatic erythrocytes (PCE) or immature erythrocytes, probably due to the inhibition of cell proliferation induced by cisplatin, which was confirmed by the nuclear division index (NDI) in bone marrow. A decrease in the ratio PCEs/NCEs (NDI) in bone marrow indicates cellular toxicity with myelosuppression [24,28]. In our study, CIS presented a significant reduction in NDI as compared to C (p < 0.05), which indicates the

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occurrence of myelosuppression in CIS. The DB + CIS group showed significant reduction in the number of micronuclei and higher NDI when compared to CIS (p < 0.05), suggesting that diabetes interferes with the genotoxic and cytotoxic activities of cisplatin diminishing its antitumor activity. No significant differences were observed between C and DB for micronuclei or NDI. The values of NDI are presented in Table 3, the photomicrographs of erythrocytes are presented in Fig. 9A–D and the quantitative analysis is presented in Fig. 9E.

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3.13. Expression of caspase-3

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The expression of caspase-3 increased significantly in CIS (19.60 ± 1.66) as compared to C (2.20 ± 0.15); whereas in DB + CIS (2.86 ± 0.32) it was significantly reduced in relation to CIS

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Fig. 10. Expression of caspase-3. (A–D) Representative photomicrographs of caspase-3 immunohistochemistry in renal tissue (400 magnification). (A) Control; (B) CIS; (C) DB and (D) CIS + DB. (E) Graphical representation of quantitative analysis. Values are expressed as mean ± SEM. C, control; CIS, cisplatin group; DB, diabetic group and DB + CIS, diabetic group treated with cisplatin. ⁄Significantly different from control (p < 0.05); #significantly different from CIS (p < 0.05). Experimental conditions are described in Section 2.

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(19.60 ± 1.66). No significant difference was observed between C (2.20 ± 0.15) and DB (1.74 ± 0.17). Photomicrographs are presented in Fig. 10A–D and the quantitative analysis is presented in Fig. 10E.

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3.14. Expression of Bcl-2

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The expression of the antiapoptotic protein Bcl-2 increased significantly in CIS (29.37 ± 2.38) as compared to C (1.14 ± 0.15); while it decreased significantly in DB + CIS (3.00 ± 0.58) as compared to CIS (29.37 ± 2.38). No significant difference was observed between C (1.14 ± 0.15) and DB (2.02 ± 0.42). Photomicrographs are presented in Fig. 11A–D and the quantitative analysis is presented in Fig. 11E.

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3.15. TUNEL assay

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The CIS group (57.75 ± 7.88) showed a significant increase in the number of TUNEL positive cells in the renal cortex and medulla compared to group C (1.37 ± 0.60). The CIS + DB group (4.63 ± 1.43)

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showed a significant reduction in TUNEL positive cells when compared to CIS (57.75 ± 7.88) indicating that diabetes decreases cisplatin-induced apoptosis in renal tissue. There was no significant difference between groups C (1.37 ± 0.60) and DB (1.80 ± 0.86). Photomicrographs are presented in Fig. 12A–D and the quantitative analysis is presented in Fig. 12E.

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4. Discussion

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Treatment of cancer in diabetic patients presents a unique challenge to clinicians. Additionally, these patients might not be properly addressed in clinical studies, and the information on how diabetes affects cancer treatment is limited [23,54,52]. It is known that diabetes attenuates the nephrotoxicity induced by cisplatin in animal models and that this attenuation is dependent on the severity of the diabetic state [66]. However, it is unclear if diabetes interferes in the antitumor activity of cisplatin. Therefore, we established a mouse model bearing: (i) diabetes, (ii) Sarcoma-180 and (iii) cisplatin-induced nephrotoxicity, in order to study the

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Fig. 11. Expression of Bcl-2. (A–D) Representative photomicrographs of Bcl-2 immunohistochemistry in renal tissue. (A) Control; (B) CIS; (C) DB and (D) CIS + DB. 400 magnification. (E) Graphical representation of quantitative analysis. Values are expressed as mean ± SEM. ⁄Significantly different from control (p < 0.05); #significantly different from CIS (p < 0.05). Experimental conditions are described in Section 2. C, control; CIS, cisplatin group; DB, diabetic group and DB + CIS, diabetic group treated with cisplatin.

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mechanisms by which diabetes attenuates the nephrotoxicity of cisplatin and if these mechanisms interfere in the antitumor activity of the drug. To our knowledge this is the first study to use an animal model with these three conditions to address these issues. The mechanism by which cisplatin causes interstitial tubule injury has not been fully elucidated [81,73], but it is known that the damage caused in kidney is proportional to the dose [53]. The dose used in this study was high enough to cause extensive damage involving more than 75% of the total area of the cortex, as revealed by histopathology. Diabetes was induced with a single administration of 150 mg of streptozotocin/kg in mice, a dose that did not cause renal damage, as confirmed by BUN and plasma creatinine of the DB group (diabetic mice not treated with cisplatin). Studies show that high doses of streptozotocin can cause nonspecific toxicity, affecting different organs, including the kidneys, while low doses affect only the pancreatic beta cells, inducing type 1 diabetes by reducing insulin production and leading to hyperglycemia [75,79].

Our previous studies with mitochondria isolated from renal cortex of rats treated with cisplatin [59,60], showed that cisplatininduced nephrotoxicity was associated with mitochondrial lipid peroxidation, GSH and NADPH depletion, markers of oxidative stress. In the present study, these markers were evaluated in homogenates of the renal cortex and only lipid peroxidation and GSH depletion were induced by cisplatin, with no significant change in the concentration of NADPH. It is known that there is an important correlation between homeostasis of GSH and NADPH as both participate in the defense against reactive oxygen species (ROS) generated mainly in mitochondria, the main cellular site generator of ROS [8]. Therefore, this difference between the findings in mitochondria and in the kidney homogenate could be attributed to the greater oxidative stress generated in mitochondria, capable of oxidizing the mitochondrial GSH and consequently the mitochondrial NADPH, which is consumed by glutathione reductase to restore the stocks of GSH from its oxidized form (GSSG). Additionally, it is known that cisplatin generates electro-


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Fig. 12. TUNEL assay. (A–D) Representative photomicrographs showing TUNEL-positive cells in renal tissue (400 magnification). (A) Control; (B) DB; (C) CIS (D) and (D) DB + CIS. (E) Graphical representation of quantitative analysis. Values are expressed as mean ± SEM. ⁄Significantly different from control (p < 0.05); #significantly different from CIS (p < 0.05). Experimental conditions are described in Section 2. C, control; CIS, cisplatin group; DB, diabetic group and DB + CIS, diabetic group treated with cisplatin.

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philic intermediates capable of reacting with SH groups (nucleophiles), which would explain the depletion of GSH without formation of GSSG and consequently, without NADPH depletion. A predominance of the first mechanism (oxidation of NADPH and GSH) in mitochondria and the second one (formation of GSH-cisplatin adducts) in the whole cell could explain these results. The DB group presented depletion of GSH but not lipid peroxidation, which suggests that the cellular redox state was already altered but the oxidation of macromolecules had not started yet. Contrarily, a study reported a significantly increased lipid peroxidation and a markedly diminished GSH in the liver of streptozotocin diabetic rats [37]. Another study reported increased formation of lipid peroxides but unchanged GSH content in the kidneys of streptozotocin diabetic rats [7]. These apparent conflicting findings might be due to the dynamics of the process, which depends on the severity of the insult. The insult triggers a sequence of biochemical events: first, reactive oxygen species (ROS) and free radicals are generated leading to decrease of GSH which will alter the cellular redox state and progress until a pro-oxidant state is established, favoring the

oxidative damage to macromolecules, such as lipids [2,51]. Therefore, depending on the conditions of the experiment, the findings might not be parallel, i.e., GSH is already decreased but not sufficiently to allow the oxidative damage to lipids and other macromolecules. Although kidneys are the main site of accumulation of cisplatin, we found high concentration of platinum in the liver. This effect could be attributed to the high dose of cisplatin used in our studies because it is known that high doses of cisplatin are associated with other toxicities, mainly hepatotoxicity [16,19]. Our findings are in line with other studies that suggest that attenuation of cisplatininduced nephrotoxicity in diabetic rats is associated with decreased renal accumulation of platinum, which in turn has been attributed in part to deficiencies in the active transport of the drug [10,62]. The lower renal accumulation of cisplatin and other nephrotoxic agents appears to be associated with the diuretic glycosuria characteristic of diabetes [67] and/or changes in the bioavailability of the drug [61]. A study in rats with diabetes experimentally induced by streptozotocin suggested that the renal organic cation


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transporter (OCT1 and OCT2) might be altered due to glycosylation of one or more of its components [27]. Our study is the first to demonstrate diabetes-induced change in the distribution of cisplatin not only in the kidney but also in other organs and especially in the tumor. Additionally, tumor remission was lower in the DB + CIS group. Taken together, these findings suggest that the mechanism of renal protection exerted by diabetes likely involves changes in the biodistribution of cisplatin, compromising its anticancer activity. The impairment of the antitumor activity of cisplatin induced by diabetes had not been previously demonstrated. The micronucleus assay detects the genotoxic activity of chemical and physical agents. The genotoxic activity of cisplatin is attributed to intrastrand and interstrand cross-links with purine bases of DNA, with consequent formation of DNA-cisplatin adducts and distortion of the DNA double helix [56,39,77]. This distortion interferes with the normal mechanisms of DNA transcription and replication, therefore inhibiting cell proliferation and causing cell death (Attia; [68]. One potentially important way by which cisplatin-DNA adducts may kill cells is by induction of programmed cell death or apoptosis [20,26]. Although the genotoxicity of cisplatin also damages healthy cells, its effects on tumor cells are more pronounced, since these cells are in the process of uncontrolled cell division. Thus, although other mechanisms are involved in the cytotoxicity of cisplatin, particularly those related to the production of reactive oxygen species (ROS), it is believed that the genotoxic action is the main mechanism involved in the antitumor action of cisplatin and that ROS are mainly responsible for the toxicity in healthy tissues, particularly in kidneys [34,60,4]. In our study, the genotoxicity of cisplatin was demonstrated by the significantly increased frequency of micronuclei (MN) in the CIS group as compared to the control group, 24 h after the administration of cisplatin. After 72 h of cisplatin administration, it was not possible to quantify the number of polychromatic (PCE) or immature erythrocytes, probably due to the severe inhibition of cell proliferation induced by cisplatin, as demonstrated by the nuclear division index (NDI) in bone marrow. NDI corresponds to polychromatic/normochromatic erythrocytes ratio (PCEs/NCEs) and its decrease is associated with toxicity in bone marrow cells and myelosuppression [24]. In our study, CIS showed significant reduction in NDI as compared to control, which is indicative of myelosuppression [28]. The DB + CIS group showed a significant reduction in the frequency of micronuclei and in NDI (myelosuppression) when compared to CIS, which indicates that diabetes interferes with genotoxicity/cytotoxicity of cisplatin and contributes to reduce the efficiency of its antitumor activity. Apoptosis is an important mechanism of cell death associated with the renal damage induced by cisplatin [17,29,77]. In our study, the characterization of apoptosis was performed by TUNEL assay and evaluation of the molecular biomarkers, caspase-3 and Bcl-2. Caspase-3 plays a crucial role in apoptosis being responsible for the activation of DNA endonucleases, cleavage and fragmentation of DNA and formation of apoptotic DNA [45]. Our research group [59] and other authors [35] have demonstrated increased activity/ expression of caspase-3 in kidneys of rats treated with cisplatin. Our present findings confirm the induction of apoptosis by cisplatin in renal cells and indicate that diabetes protected against this effect in this experimental model, which is in line with the nephroprotective effect of diabetes indicated by other studies [66,62]. The anti-apoptotic protein Bcl-2 plays an important role in apoptosis regulation, both in physiological and pathological conditions [6,71]. Bcl-2 prevents the loss of mitochondrial membrane potential, release of cytochrome c into the cytosol and consequently, the activation of caspases by the mitochondrial pathway [14,71,15,76]. It is known that overexpression of Bcl-2 inhibits the activation of caspase-3 [55], but in our experimental model we observed increased expression of both proteins in the CIS group

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as compared to control. The apparently contradictory increase of both Bcl-2 (anti-apoptotic) and caspase-3 (effector of apoptosis) could be attributed to a temporary cell response to the activation of caspase-3, as an attempt to counteract the apoptotic process triggered by cisplatin. A study to evaluate the nephroprotective potential of sildenafil in rats treated with cisplatin also reported increased expression of caspase-3 and Bcl-2, but after 96 h of cisplatin treatment [43]. In our study, the increase in Bcl-2 was higher than in the previous study and was observed 72 h after cisplatin treatment. The DB + CIS group had a significant reduction in the expression of Bcl-2 as compared to the CIS group. Since caspase3 was significantly lower in DB + CIS than in control, this result is in line with our hypothesis that the increase of Bcl-2 would be a cellular response to counteract the apoptosis already initiated. The detection of apoptosis by TUNEL assay confirms caspase-3 results and the morphological alterations found, and it is also in line with previous studies in different experimental models [82,43].

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5. Conclusions

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The experimental model is suitable for the study of the antitumor activity of cisplatin in diabetic animals and might be adjusted to evaluate the effects of diabetes on other drugs. Diabetes decreases lipid peroxidation, apoptosis and the histopathological changes induced by cisplatin in kidneys, but has no effect on GSH depletion. It also decreases platinum accumulation in multiple organs and tumor, decreases tumor remission, survival rate (in cisplatin-treated animals) and the genotoxicity of cisplatin. This is the first study to demonstrate that diabetes-induced protection from cisplatin nephrotoxicity is associated with impairment of the antitumor activity of the drug.

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Conflicts of Interest

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The authors declare no conflicts of interest.

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Transparency Document

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The Transparency document associated with this article can be found in the online version.

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6. Uncited reference Attia [3].

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Acknowledgments

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The authors would like to thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Grant Number 2009/11938-4) for the financial support. The authors would also like to thank Professor José Roberto Mineo, Ph.D. (Immunology, Federal University of Uberlandia, MG, Brazil) who kindly provided Sarcoma 180 cells.

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Evaluation of the protective effect of Cystone against cisplatin-induced nephrotoxicity in cancer patients, and its influence on cisplatin antitumor activity.

Tropisetron attenuates cisplatin-induced nephrotoxicity in mice.

Renal protective effect of selenium on cisplatin-induced nephrotoxicity.

Effect of infliximab against cisplatin-induced nephrotoxicity.

Ameliorative Effect of Daidzein on Cisplatin-Induced Nephrotoxicity in Mice via Modulation of Inflammation, Oxidative Stress, and Cell Death.

Biodistribution and antitumor effect of Cetuximab-targeted lentivirus.

Prevention of cisplatin nephrotoxicity.

Protective effects of C-type natriuretic peptide on cisplatin-induced nephrotoxicity in mice.

Brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity.

Neutralizing effect of sodium thiosulfate on antitumor efficacy of cisplatin for human carcinoma xenografts in nude mice.

Effect of aqueous extract of Rheum ribes on cisplatin-induced nephrotoxicity in rat.

Effect of creatine and pioglitazone on Hk-2 cell line cisplatin nephrotoxicity.

Effect of pomegranate flower extract on cisplatin-induced nephrotoxicity in rats.

Effect of testosterone on Cisplatin-induced nephrotoxicity in surgically castrated rats.

Partial reversibility of cisplatin nephrotoxicity in children.

Significance of Tamm-Horsfall protein excretion in diabetes mellitus and cisplatin nephrotoxicity.

Transgenic expression of the human MRP2 transporter reduces cisplatin accumulation and nephrotoxicity in Mrp2-null mice.

Mitochondrial dysregulation and protection in cisplatin nephrotoxicity.

Influence of high-energy shock waves and cisplatin on antitumor effect in murine bladder cancer.

LY2109761 enhances cisplatin antitumor activity in ovarian cancer cells.

Ginsenoside Rg5 Ameliorates Cisplatin-Induced Nephrotoxicity in Mice through Inhibition of Inflammation, Oxidative Stress, and Apoptosis.

Preventive Effect of Dihydromyricetin against Cisplatin-Induced Nephrotoxicity In Vitro and In Vivo.

Comparative study of cisplatin and carboplatin on pharmacokinetics, nephrotoxicity and effect on renal nuclear DNA synthesis in rats.

Protective effect of selenium on cisplatin induced nephrotoxicity: A double-blind controlled randomized clinical trial.