The experimental diets contained the same amounts of nutrients, but included different fatty acid compositions (Table I). In brief, the MA and control (CTR) diets were modifications of the AIN-76 diet. The MA diet contained 5 or 10% SUNTGM33, which in turn contains 48% MA (12,13). SUNTGM33 is a microbial oil obtained by fungal fermentation (16). The CTR diets contained 5 or 10% olive oil (Nakalai Tesque), which contained 74.7% oleic acid (OA). OA is a precursor of MA. The composition of SUNTGM33 and olive oil have been described in our previous studies (12,13). The experimental diets contained 2.4 or 4.8% MA, while the CTR diet did not contain MA. The concentration of MA in the experimental diet was consistent with our previous studies (12,13). The highest concentration of MA (4.8%) in the diet contained almost the same amount of MA, which was considered the upper limit on the blend material level for the diet used in rat feeding studies.

DMBA was obtained in powder form from Eastman Chemical. Prior to its use, DMBA was dissolved in sesame oil at 120˚C (DMBA 1,000 mg/50 ml sesame oil). A single dose of 80 mg/kg body weight was administered orally (17). The same amount of sesame oil without DMBA was administered to the animals in the CTR group.

The study protocol and animal procedures were approved by the Animal Care and Use Committee of Kansai Medical University, Hirakata, Osaka, Japan (permit no. 13-060). Throughout the experiments, the animals were housed and treated in accordance with the Guidelines for Animal Experimentation of Kansai Medical University. In the present study, the following criteria for humane endpoints were also used (NIH guidelines for endpoints in animal study proposals): i) A tumor burden >10% of the animal body weight; ii) the tumor should not exceed 40 mm in any one dimension; iii) tumors that become ulcerated, necrotic or infected; iv) tumors that interfere with the eating ability or impair the ambulation of the animals.

In brief, 86 female Sprague-Dawley rats [Crl:CD(SD), 6 weeks old] were purchased from Charles River Laboratories Japan. They were housed in groups of 4 or 5 in plastic cages with paper bedding (Paper Clean; Japan SLC, Inc.) in a specific pathogen-free environment maintained at 22±2˚C and at 60±10% relative humidity with a 12-h light/dark cycle (lights on at 8:00 a.m. and lights off at 8:00 p.m.). In the experiment with 2.4% MA, the rats were randomly divided into 4 groups as follows: The CTR + sesame oil (n=5), CTR + DMBA (n=13), 2.4% MA + sesame oil (n=5) and 2.4% MA + DMBA (n=13) groups. In the experiment with 4.8% MA, the rats were divided into 4 groups as follows: CTR + sesame oil (n=10), 4.8% MA + sesame oil (n=10), CTR + DMBA (n=15) and 4.8% MA + DMBA (n=15) groups.

Fresh sterilized stocks of the pellet diet were provided to the animals twice a week starting at 6 weeks of age. The previous pellets were discarded to minimize the ingestion of oxidized fatty acids. The animals in the experimental groups received DMBA, whereas the animals in the CTR groups received sesame oil at 7 weeks of age and all animals remained on the same diets for the remaining duration of the experiments (until 19 weeks of age). The experimental diets and water were available freely. During the dosing period, the dose of the diet ingested, body weight and tumor volume were measured once a week. The tumor volume was calculated using the standard formula: Width² x length x 0.5. The tumor volume measurement was used for monitoring of tumor incidence and growth. Prior to sacrifice, all rats were anesthetized with isoflurane (Wako Pure Chemical Industries, Ltd.). Before necropsy, the isoflurane was made to soak into paper and was put in closed chamber. Subsequently, rats were anesthetized in a pervasive chamber of isoflurane which was vaporized. A total of 4% of isoflurane was used for the induction of anesthesia and blood was sampled by inferior vena cava puncture. Subsequently, the animals were sacrificed by exsanguination and aortic transection. At necropsy, all organs were examined macroscopically and the breast tissue and tumors were examined histologically. The tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin and finally stained with hematoxylin and eosin (Wako Pure Chemical Industries, Ltd.). Cell kinetics were also assessed. The serum samples and the sections of the non-tumor breast tissues were used for fatty acid analysis. During the examination of the animals receiving the 4.8% MA diet, fatty acid analysis was not carried out.

The cell kinetics (cell proliferation activity and apoptosis) in the 6 largest DMBA-induced tumors were evaluated. The cell proliferative activity was evaluated using anti-Ki-67 antibody (cat. no. 418071, prediluted, clone SP6, Nichirei Biosciences). The incubation condition was 1 h at room temperature. The induction of apoptosis was evaluated by the anti-phospho-histone H2A.X (γ-H2A.X) antibody (cat. no. 2577S, x100, clone Ser139, 1:100; Cell Signaling Technology, Inc.), an immunomarker of the DNA damage response. The incubation condition was 1 h at room temperature. Immunohistochemical analysis was performed with the Histofine MAX-PO for rats kit (Nichirei Biosciences) according to the manufacturer's protocol. Each slide was scanned with a high-resolution digital scanner (NanoZoomer 2.0 Digital Pathology; Hamamatsu Photonics) to prepare the digital images (NDPI image). The NDPI image files were opened in color mode with the NDP.view software (Hamamatsu Photonics). The images were converted to the JPEG files (magnification, x400) in 5 randomly selected areas within each tumor and were analyzed by immunohistochemical staining, as previously described (12,18,19). The Ki-67 and γ-H2A.X labeling indices were assessed by positive cells/1,000 cells as an index of cell kinetics.

The fatty acid composition of the total phospholipid fraction of serum was determined and mammary gland samples were extracted using the method described in the study by Bligh and Dyer (20). The total phospholipid fraction was separated by thin-layer chromatography. The compound 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids, Inc.) was added as an internal standard. Total phospholipid fractions were transmethylated with HCL-methanol and subsequently, the fatty acid composition was analyzed by gas chromatography (GC-2014, Shimadzu Corporation) with a capillary column DB-225 (0.25 mm x 30 m x 0.25 µm) (J&M Scientific, Folsom). The system was controlled with the gas chromatography software (GC solution; Shimadzu Corporation). The fatty acid composition of the total lipid fraction of the non-tumor mammary gland was determined. Frozen tissues were thawed, minced and homogenized 3 times for 10 sec in 8 ml chloroform-methanol (2:1) by a polytron homogenizer (Kinematica). The fatty acid analysis of the total lipid content in the breast tissues was performed by a similar method as that described in the analysis of the serum and mammary glands, with the exception of excluding the separation step performed by thin-layer chromatography (9,10,20).

The values are expressed as the means ± standard error of the mean. The parameters body weight, tumor volume, tumor weight, fatty acid composition and the percentage of Ki-67-positive and γ-H2A.X-positive cells among the groups were analyzed using the Student's t-test. The incidence of breast cancer was analyzed using a χ² test.

During the dosing period, the daily dose of food ingestion was compatible among the different groups. The parameter body weight did not reveal significant differences when the 2.4% MA diet was used, while in the 4.8% MA diet experimental protocol, the body weight in the group administered the 4.8% MA diet and exposed to DMBA was significantly decreased compared with that of the group administered the 4.8% MA diet and given sesame oil (Fig. 1).

All mammary tumors were examined and confirmed histologically as mammary cancers. At the end of the experimental period, although the tumor incidence in both the 2.4 and 4.8% MA diet groups was lower than that noted in the CTR diet groups, the differences were not significant (Fig. 2). The mean values of DMBA-induced breast tumor weight in the CTR diet and 2.4% MA diet groups were 1,323±251.4 mg and 1,019.3±178.6 mg, respectively, while the final average breast tumor weight in the CTR diet and 4.8% MA diet groups was 941.3±231.4 mg and 1,235.6±208.4 mg, respectively. Neither of these groups demonstrated a significant difference (Table II and Fig. 3). The mean values for tumor volume in the CTR diet and 2.4% MA diet groups were 1,582±451 mm³ (maximum diameter, 29 mm) and 1,520 ± 397 mm³ (maximum diameter, 31 mm), respectively, while the final average values for tumor volume in the CTR diet and 4.8% MA diet groups were 633±1301 mm³ (maximum diameter, 27 mm) and 930±2,418 mm³ (maximum diameter, 32 mm), respectively. Neither of these groups demonstrated a significant difference. The largest volume in the CTR diet and 2.4% diet groups were 7,406 mm³ (28 mm in diameter) and 10,478 mm³ (31 mm in diameter), respectively, while the largest tumor volumes in the CTR diet group and 4.8% MA diet groups were 5,832 mm³ (27 mm in diameter) and 10,240 mm³ (32 mm in diameter) respectively. The mean values of DMBA-induced breast tumor multiplicity in the CTR diet and 2.4% MA diet groups were 5.0±1.3 and 4.1±0.3, respectively, while the final average breast tumor multiplicity values in the CTR diet and 4.8% MA diet groups were 2.3±0.7 and 2.0±0.3, respectively. Neither of these groups demonstrated a significant difference (data not shown).

In the groups in which DMBA was not administered, the presence of breast tumors was not observed, in the presence of either the CTR or MA diet in both experimental settings. No conspicuous morphological differences were noted between the CTR diet and the MA diet groups. No lymph node metastasis was noted in any animal.

The percentages of Ki-67-positive cells and γ-H2A.X-positive cells from the CTR diet and MA diet groups were compared with regard to the ratio of proliferative cells and the apoptotic cell number. The proliferative cell number and apoptotic cell ratio are presented in Table III and Fig. 4. Although the MA diet exhibited a tendency to suppress cancer cell proliferation, the differences observed were not significant. In both experimental settings with the 2.4% MA and 4.8% MA diet, the ratio of apoptotic cells was exactly the same.

The different diet groups exhibited different fatty acid compositions in serum and mammary tissues, reflecting the content of the respective diets. Exposure to DMBA did not affect the fatty acid composition. The n-3, n-6 and n-9 (MA) fatty acid composition levels in the serum of the animals receiving the 2.4% MA diet were significantly increased compared with those noted in the CTR diet group, whereas the concentrations of OA, LA, AA and DHA were significantly decreased compared with those noted in the CTR + sesame oil group (Fig. 5A). The levels of OA and LA in the non-tumor mammary gland were significantly decreased and the level of AA in non-tumor mammary gland was significantly increased in the MA group compared with those noted in the CTR + sesame oil group (Fig. 5B). The changes in serum fatty acid composition resulted in a significant decrease in the N-6/N-3 ratio in the 2.4% MA diet group (Fig. 6A). However, the N-6/N-3 ratio noted in the non-tumor breast tissue did not exhibit any marked changes (Fig. 6B).