Research and Publication Ethics: The study procedures were approved by the Ethics Review Committee of Firat University, Elazig, Turkey (Protocol No: 2016/57) and complied with the Animal Welfare Act guidelines.
Animal Design: Female Wistar Albino rats were supplied by the Firat University Experimental Research Center. All animals were maintained on a regular dark-light cycle (12-h light–dark cycle), with free access to pellet diet and water ad libitum during the whole experimental duration. All animals were housed in clean polypropylene cages (four rats/cage) under controlled indoor temperature (2±2°C) and humidity (44±5%) conditions.
Experimental Setup: Thirty-two healthy female rats were separated into 4 groups. Group I was given a single dose of 80 mg/kg of DMBA dissolved in 1 mL olive oil by gastric gavage on the 15th day of the beginning of experimental period. Breast cancer was allowed for 90 days to monitor the onset of tumorigenesis. Group II, III and IV received DMBA as in Group I and after 90 days, Group II was given 3 mg/kg RAL (dissolved in 1 mL DMSO) by gavage 3 times a week for 6 weeks. Group III rats were given FLX dissolved in 1 mL of distilled water at a dose of 30 mg/kg by oral gavage daily for 6 weeks. Group IV were simultaneously treated by FLX plus RAL at the doses indicated above. Rats were palpated every week to monitor the onset of tumorigenesis.
Biochemical Analysis:
Preparation of Mammary Tissue Homogenate: Homogenate of breast tissues was prepared Phosphate Buffered Saline (PBS) (0,1 M, pH: 7.4) using an automatic tissue homogenizer machine (Ultra TurraxType T25-B, IKA Labortechnic, Germany). Lowry method was preffered to measure total protein contents 11. In an alkaline medium, the copper ion (Cu+2) forms a complex with peptide bonds in proteins and reduced to Cu+1. Reduced copper and the amino acids Tyr, Trp and Cys in the side chain of proteins reduce the Folin-Phenol reagent and cause deep blue color formation. The intensity of the resulting color is measured spectrophotometrically at 660 nm.
Determination of MDA Levels: The levels of MDA were estimated by the thiobarbituric acid test according to the process described by Ohkawa et al. 12. This method is based on a condensation reaction of two molecules of TBA with one molecule of MDA to give a pink colored compound which can be measured at 532 nm wavelength.
Assay for Mammary Antioxidant Enzymes Activities: Superoxide dismutase enzyme was determined by the method modified by Sun et al. 13. In this method, superoxide radical is produced by xanthine-xanthine oxidase system and the resulting superoxide radical reacts with iodonitrotetrazolium (INT) to form violet colored formazone dye and this color intensity is measured at a wavelength of 505 nm. This reaction is inhibited and % inhibition is calculated due to CuZn-SOD activity in the medium. CuZn-SOD activity is expressed as U/g protein for tissue.
In the GPx activity assay, reduced glutathione (GSH) in the presence of H2O2 is oxidized by GPx to oxidized glutathione (GSSG). Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is used when oxidized GSSG is converted back into GSH by glutathione reductase enzyme. The amount of NADPH used is monitored at 340 nm wavelength as a decrease in absorbance 14.
CAT activity was measured in tissue homogenate at 25ºC by the method specified by Aebi 15. Here, the decomposition rate of substrate H2O2 was monitored spectrophotometrically at 240 nm for 30 seconds.
Assessment of CA 15-3 Levels: The tissue CA 15-3 levels were measured by using Enzyme-Linked Immuno Sorbent Assay (ELISA) for rats (Cat No: YLA0753RA, YL Biont, Shanghai, China, YL Biotech Co, Ltd.) method. A 96 well microplate, pre-coated with an antibody specific for CA 15-3, was provided by the commercial kit. The assay procedure was summarized as follows: standards and samples are pipetted into the wells and then added a Horseradish Peroxidase (HRP) conjugated antibody specific for CA 15-3. To remove any unbound reagent, washing process was carried out. Following a substrate solution was added to the whole wells, the color developed in proportion to the amount of CA 15-3 bound in the initial step was observed. The results were calculated by comparing the absorbance of the samples to the standard curve by ELISA reader.
Statistical Analysis: All data were analyzed using SPSS 22.0 statistical software package (IBM, Armonk, NY, USA). One-way analysis of variance (ANOVA) followed by post hoc Tukey HSD was used to assess difference between the groups. All the results are reported as mean±standard deviation. P<0.05 were considered as significant.