In contrast to the normal rat mammary tissues, the abdominal-inguinal and contralateral mammary glands of rats induced with breast cancer showed alteration in their tissue structures (Figure 1), which in comparison with the report by Murray et al. [18], was similar to a pregnant or lactating mammary gland phenotype. A number of alterations were observed in the morphology of breast tissues in the group of rats injected with N-methyl-N-nitrosourea (NMU). An increase in the number of acini, further epithelial differentiation with secretory material, ductal carcinoma in situ (DCIS), hyperplastic ducts containing more than three layers of epithelial cells and large dilated ducts in a lobular configuration were also noticed. There were occasional areas showing benign proliferation of the ducts and crowding of acini. In one of the rat samples, scattered atypical cells with hyperchromatic nuclei (an indication of early stromal invasion), in combination with DCIS was noted. This is an indication of early invasive cancerous change although there was no significant alteration that was noticed in the stroma.

Sagittal T1 Fat Sat contrast enhanced image of the rat was successfully scanned using gadolinium as a contrast agent at 14 weeks after injection of NMU (Figure 2). There was marked enhancement of the upper set of the mammary glands in the post gadolinium images measuring 2 mm in the long axis but no noticeable enhancement in the lower mammary glands.

When proteins from control rat serum samples were separated by 2-DE and stained with silver, complex protein profiles containing hundreds of spots were obtained. Serum samples of each treated rat group were subsequently resolved using the same technique. Figure 3 shows the representative serum protein profiles of normal female control rats and rats treated with NMU and LPS, respectively. Identities of the 2-DE resolved proteins from serum isolated in the first (control samples) and the final (rats assumed to have cancer/inflammation) weeks were initially established by visual comparison of the 2-DE gels with previously published protein maps obtained from rat sera [19]. Identities of the serum proteins were then confirmed by mass spectrometry and database search (Table 1).

Volume analysis of the 13 spot clusters within and between the different groups of rat serum samples demonstrated comparable expression of almost all the highly abundant proteins detected. For most protein clusters, comparable expression was detected between the groups of rats treated with NMU compared with those of the rat normal serum samples (Figure 4a). The spot clusters of albumin and fetuin-B precursor were apparently not conducive to the analysis. In contrast, the group of rats injected with LPS showed significant variation in the expression of alpha-1B glycoprotein (Figure 4b).

Twelve, two month old, inbred and virgin female Sprague-Dawley rats were used. Each rat was individually caged at the animal quarters at 23–25 °C and 35–55% humidity, with normal light-dark cycle. Regular chow pellets and normal tap water were supplied ad libitum. The first bleeding was performed five days prior to treatment with chemicals and the serum samples obtained were used as normal controls. Blood was taken from the tail vein using 23 gauge butterfly needles and serum samples were stored at −80 °C. The rats were sacrificed 18 weeks after the first injection of chemicals and their breast tissues were dissected. All experimental procedures were approved by the Animal Research Committee of the University of Malaya Medical Centre in accordance with the guide for care and use of laboratory animals.

N-Methyl-N-nitrosourea (NMU) and lipopolysaccharide (LPS) from E. coli were purchased from Sigma Aldrich Chemical Company (St Louis, MO, U.S.). Six rats received a single intraperitoneal dose of 50 mg/kg NMU for inducement of breast cancer while the rest received 0.06 mg/kg LPS to initiate inflammation. The NMU solutions were used within 15 min of preparation.

The abdominal-inguinal and contralateral mammary glands were collected from euthanized rats at 18 weeks after the NMU injections. The mammary glands were fixed in 10% formalin overnight, processed and embedded into paraffin blocks. Sections were cut at 4 μm thickness and stained with Hematoxylin (Biostain Ready Reagents Ltd, Cheshire, UK) and Eosin (Richard-Allan Scientific, Kalamazoo, MI, U.S.) on frosted slides. Breast tissues were examined under a light microscope to observe for microscopic cancerous and precancerous lesions and confirmed by an experienced histopathologist.

Rats at the 14th week after NMU injections were anesthetized by intramuscular injection with ketamine (80–110 mg/kg) and xylazine (12 mg/kg) (Troy Laboratories Australia Pty Ltd, Sydney, Australia), to prevent movement prior to MRI. A 24 gauge angiocatheter was placed in the tail vein and injected with heparin-saline to prevent blockage. An IV contrast agent, gadopentetate dimeglumine (Magnevist—469 mg/mL; Bayer Schering Pharma AG, Berlin, Germany), diluted with heparinized sterile saline solution at 0.2 mL/kg, was injected at the contrast phase of the MRI. The rats were placed on a polystyrene mold that was inserted into the knee coil of a standard clinical 3 Tesla magnet GE Healthcare MRI equipment. A scout image was taken for anatomical references to ensure that the upper and lower mammary glands were covered. T1, T2, and post gadolinium fat sat T1 images were acquired in different planes (see Table 2 for parameter settings).

Although a 30-min sedation for MRI was planned for the rats, scans had to be done within 20 min as some of the rodents developed significant bradycardia and extreme low blood pressure after 15 min. This may be partly due to hypothermia because of the cold MRI room, which may be reduced by using a hot air blower that was directed at the knee coil [22,23]. In a few of the sedated rats, imaging had to be abandoned as contrast was not able to be administered due to constriction of vessels and decreased peripheral blood circulation, especially at the sites for IV contrast administration in the tail vein.

2-DE and mass spectrometry using the 4800 Plus MALDI TOF/TOF analyzer (Applied Biosystems/MDS Sciex, Toronto, Canada) were performed as previously described [8,24]. Protein spots were identified using the MASCOT search engine (Matrix Science Ltd, London, U.K.; release version 2.1.0) against rattus entries in the Swiss-Prot database (Last update October 23, 2008, containing 261513 sequences). The following variables were set as search parameters: trypsin was used; up to one missed cleavage was allowed; variable modifications included were carbamidomethylation of cysteine and oxidation of methionine; the mass tolerance for MS precursor ion and MS/MS fragment ion were 100 ppm and 0.2 Da, respectively; and only monoisotopic masses were included in the search.

The GE ImageMaster^TM 2D Platinum Software version 7 was used to analyze expression of proteins in the 2-DE profiles. Percentage volume contribution of protein spots, which refers to the spot volume of a protein expressed as a percentage of the total spot volume of all detected serum proteins, was calculated to detect proteins that were differentially expressed in the rat serum. The ANOVA statistical test was used to analyze the protein spot differences between the profiles. A p value of less than 0.05 was considered significant.