Thirty Wistar rats – gender male; age 3 months and average weight 100g - were recruited for the study and equally divided into five groups A, B, C, D, and E. The treatments given to the animals consist of anticancer drugs in combination with OA. Group A (control), received NaCl 0.9%; group B, received only oleic acid (OA) (1.5ml/rat); group C was given cyclophosphamide (CPP) (20mg/rat) + OA; group D was administered daunorrubicine (DRB) (4mg/rat) + OA; and group E was treated with dexrazoxane (DXN) (50mg/rat) + OA. Every treatment was by intraperitoneal route and the administration was every 24 h for 5 days. The animals were procured from Bioterium of Metropolitan University of Mexico City and housed six per cage in clean plastic cages and allowed to acclimatize in the room environment for 1 day. Animals were maintained in a mass air displacement room with a 12-h light:12-h dark cycle at 22 ± 2°C with a relative humidity of 50 ± 10%. Balanced food (Rodent diet 5001) and drinking water were given to the animals ad libitum. On the last day of the treatments, blood samples were obtained to measure glucose hemoglobin and triglycerides. After that, the rats were sacrificed by decapitation and their brains were extracted and sectioned into cortex, striatum, and cerebellum/medulla oblongata (CMO). We do not use anesthetics because they could alter our results. The sections were immediately immersed in NaCl at 0.9% and kept at 4°C. 3ml of tris-HCl 0. 05M pH 7.2 was employed in the homogenization of every section of the brain and used to assay the peroxidation of lipids (TBARS), H2O2, Na+, K+ ATPase activity, glutathione (GSH), and dopamine using previously validated methods. To avoid the degradation and loss of integrity of the samples, they were stored at –20°C until analyzed. Animal management and care was conducted in accordance to the international guidelines for animal care and to the Mexican Guidelines ZOO-062, and that allowed by the Laboratory of Animal Care Committee of The National Institute of Pediatrics.
Triglycerides, blood hemoglobin and glucose were measured at the end of the treatments. Glucose and triglyceride concentrations were measured with two sets of 20µl of volume non-anticoagulant tail-end blood samples using Accu- Chek glucose reactive paper (Roche Mannheim, Germany) and reported in mg/dl.
2.1.1 Measurement of Dopamine (DA)
DA concentrations were measured from a 9000 rpm 10 min centrifuged HCLO4 homogenized supernatant tissue. The centrifugation was done in a microcentrifuge (Hettich Zentrifugen, model Mikro 12-42, Germany), and the measurement was carried out based on Calderon et al [2,28] method using FL Win Lab version 4.00.02 software. The values were deduced from a previously standardized curve and reported as nM/g of wet tissue.
2.1.2 Glutathione (GSH) measurement
To measure GSH levels, the homogenized tissue supernatant obtained prior to centrifugation for 5 min period at 9000 rpm (Mikro 12-42, Germany centrifuge) using Hissin and Hilf modified method [2,29].
2.1.3 Lipid peroxidation measurement
The approach employed in the measurement of TBARS was Gutteridge and Halliwell modified technique [21]. A 1 ml of the tris-HCl0. 05M pH 7.4 brain homogenate was mixed with 2ml thiobarbaturic acid (TBA) that contains 1. 25g TBA, 40g trichloroacetic acid (TCA) and 6. 25ml of concentrated chlorhydric acid (HCL) diluted in 250ml of deionized H2O. The resulting mixture was heated to boiling point for 30min (Thermomix 1420) and cooled for 5 min in an ice bath. This was subjected to a centrifugation at 700g for 15min (Sorvall RC-5B Dupont). The floating tissue absorbances were spectrophotometrically (Helios-α de UNICAM) read in triplicate at 532nm. The concentration of reactive substances to the thiobarbaturic acid (TBARS) was expressed in µM of Malondialdehyde/g of wet tissue [30].
2.1.4 Total ATPase determination
ATPase activity was assessed based on Calderón et al method [31]. One mg (10%) w/v of the tris-HCl0. 05M pH 7.4 tissue homogenate was subjected to an incubation for 15 min in a solution containing 3mM MgCl2, 7mM KCl and 100mM NaCl. 4 mM tris-ATP was added to the mixture and the incubation was repeated for another 30 min at 37oC in a shaking water bath (Dubnoff Labconco). The reaction was detained using 100 µL 10% trichloroacetic acid w/v. The samples were final centrifuged at 100 g for 5 min at 4O °C [32]. Inorganic phosphate (Pi) was measured in duplicates using one supernatant aliquot as reported by Fiske and Subarrow [33]. The reading of the absorbance of the supernatant of each sample was carried out spectrohpotometrically at 660 nm in a Helios-α, UNICAM and expressed as mM Pi/g wet tissue per min.
2.1.5 H2O2 determination
H2O2 measurement was performed with Asru [34] and Hernandez [35] techniques after modification. The brain regions (cortex, striatum, CMO) were separately diluted in 3 ml of tris-HCl 0. 05M pH 7.4 buffer and homogenized. 100µl of the resulting homogenate of each brain region was mixed with1ml of potassium dichromate solution (K2Cr2O7). The mixture was heated to boiling point for 15min (Thermomix 1420) and cooled for 5 min in an ice bath. Then, the samples were subjected to a centrifugation at 3,000g for 5min (Sorvall RC-5B Dupont). The reading of the floating absorbances was spectrophotometrically done in triplicate at 570nm (He?ios-?, UNICAM). H2O2 concentration was expressed in µMoles [30].
2.1.6 Statistical analysis
For statistical analysis, the fisher estimation test (ANOVA) and the nonparametric test of Kruskal-Wallis were used with their corresponding contrasts and previous to variance homogeneity comparison. Statistical significance was put at p-values of <0.05 [2,36]. JMP Statistical Discovery Software, version 8 .0.0 from SAS was employed in the statistical tests [2].