The 7,12-dimethylbenz(a)anthracene (DMBA) (purity ≥95%) and tartrazine were obtained from Sigma-Aldrich® (Standford, Germany). The natural dye made from corn starch was obtained at the slaughterhouse market (Maroua, Cameroon). The anesthetics, diazepam (Valium® 10 mg/2 mL) and ketamine (Ketamine hypochloride 50 mg/mL) were obtained from Roche (Fontenay-sous-Bois, France) and Rotex Medica (Tritau, Germany), respectively. ELISA kits for determining alpha-fetoprotein, CA 15–3 and estradiol levels were obtained from Elabscience® (Willich, Germany).
Forty (40) prepubertal Wistar rats (Rattus norvegicus) aged 41 to 51 days at the start of the experiment and weighing between 70 and 85 g were obtained from the breeding facility of the Animal Physiology Laboratory of the University of Yaoundé 1. These rats were distributed 8 per group in plastic cages at room temperature in the animal house of the Department of Biological Sciences, Faculty of Sciences, University of Maroua where the study was conducted. They had free access to water and were fed with standard rat chow containing: corn meal (36.6%), bone meal (14.5%), cotton seed meal (7.3%), fish (4.8%), cooking salt (0.3%) and vegetable oil.
Housing and animal treatments were approved by the Joint Institutional Review Board Animal & Human Bioethics of the Faculty of Science (University of Yaounde 1), which adopted the directives established by the European Union on the care of animals (EEC Council 86/609).
Induction of breast cancer
Breast cancer was induced according to the method of Mefegue et al. . Briefly, 50 mg/kg BW of DMBA dissolved in 1 mL of olive oil was thoroughly sonicated and injected subcutaneously (s.c) onto the right inguinal mammary gland of the pubescent rats (55–60 days) to induce mammary tumors. Alternatively, normal animals were given olive oil only.
Treatment of animals
The dose of tartrazine used in this study was derived from the work of Das and Mukherjee , who showed that tartrazine is non-mutagenic and non-genotoxic at doses of 50, 100 and 200 mg/kg BW. The smallest safe dose (50 mg/kg) was therefore chosen to assess its possible promoter effects on the occurrence of breast cancer in female rats exposed to DMBA. For this to be done, 40 female Wistar rats aged 41 to 51 days were acclimatized for 07 days, afterward the rats were randomly assigned into five groups of 8 animals each as follows: Two normal control groups given distilled water (NOR) or tartrazine (NOR + TARZ), respectively. The other 3 groups were exposed to DMBA and received distilled water (DMBA), tartrazine (DMBA + TARZ) and a natural corn starch dye (DMBA + COL). Treatment was performed by gavage a week before exposure to DMBA and thereafter, it continued for 20 weeks. The animals were weighed weekly and palpated twice a week to detect tumor. The moribund rats were sacrificed under anesthesia and for those which died during the experiment autopsy were performed and all parameters have been recorded. At the end of treatment, all the surviving animals were fasted for 12 h, weighed and sacrificed by decapitation under anesthesia consisting of a mixture of ketamine (10 mg/kg BW, i.p.) and diazepam (50 mg/kg BW, i.p.).
Blood was collected in dry tubes and centrifuged at 3000 rpm for 15 min, then stored at 4 °C for subsequent biochemical analyzes. The skin was then dissected to expose the breast tumors which were all removed, counted and weighed. Estrogen target organs (ovaries, uterus, vagina and mammary glands), major breast cancer metastasizing organs (femur, brain, liver and lungs) and certain organs of interest for toxicity studies (spleen, kidneys and adrenal glands) were removed and weighed. All organs were immediately fixed in 10% formalin for histological analysis.
The tumor incidence (percentage of affected rats per group), relative tumor weight (tumor weight divided per animal body weight) and tumor burden (the cumulative tumor weight of animals in a group) were determined. The tumor size was measured using an electronic caliper (IGAGING®) and tumor volume was calculated using the formula of Kubatka et al. : width 2 × length/2.
Preparation of homogenates of mammary glands and tumors
A part of each mammary gland or tumor was cut, weighed and crushed using Potter teflon-glass on ice using sodium phosphate buffer (0.1 M; pH 7.5) to afford a 20% final homogenate. After centrifugation at 3000 rpm for 15 min at 4 °C, the supernatant was stored at − 20 °C for the determination of total protein level and oxidative status in mammary gland/tumor.
Biochemical and hematological analysis
Different hematological parameters were evaluated using a MINDRAY BC-2800 Auto Hematology Analyzer from Shenzhen Mindray Bop-medical Electronics Co., Ltd. These parameters included: white blood cell (WBC) count, lymphocytes, monocytes, granulocytes, red blood cell (RBC) count, hematocrit, hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and platelets.
The quantitative determination of estradiol, Alpha-fetoprotein (AFP) and breast cancer biomarker CA 15–3 levels in sera was done using enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s instructions (Monobind Inc.®, California, USA). The estimation of the total protein levels was performed following the methods described by Gonal et al. . Oxidative stress parameters likesuperoxide dismutase (SOD) activity, malondialdehyde (MDA) level, catalase activity and GSH level were measured following the methods of Misra , Wilbur et al. , Sinha  and Ellman , respectively.
Histopathological changes in mammary glands and tumors were determined by using 5-μm tissue sections of paraffin-embedded organs stained with hematoxylin and eosin. The images were captured using the complete Zeiss equipment consisting of a microscope Axioskop 40 connected to a computer where the images were transferred and analyzed with the MRGrab 1.0 and Axio Vision 3.1 softwares, all provided by Zeiss (Hallbermoos, Germany). The breast tumors were classified using the histopathologic criteria from Russo and Russo .
Analysis of variance (ANOVA) followed by Dunnett’s post test for multiple comparisons were used for the various statistical analyzes using GraphPad Prism version 5.00 software. All the animals were included in the analysis and comparison was made between different control and treated groups. The data obtained were expressed as the mean ± standard error of the mean (SEM) and the difference was considered significant at a probability level of 5% (p < 0.05).
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