Fish oil is known to protect from many types of cancers of the colon, liver, breast, prostate and lung [1–3]. The objective of the present study was to evaluate the role of fish oil [Maxepa, supplemented at a dose of 0.5 ml is equivalent to 90 mg eicosapentaenoic acid (EPA) and 60 mg docosahexaenoic acid (DHA)] on cell proliferation, expression of p53 tumor suppressor protein and DNA protein crosslinks (DPCs) in a defined model of chemical rat mammary carcinogenesis. Mammary carcinogenesis was initiated by a single, intravenous (i.v.) tail vein injection of 7,12 dimethylbenz(α)anthracene (DMBA) at a dose of 5 mg DMBA/2 ml corn oil/kg body weight in female Sprague-Dawley rats at 7 weeks of age. Fish oil supplementation was started daily, 2 weeks prior to DMBA injection and continued for 24 (31 weeks of animal age) weeks and 35 (42 weeks of animal age) weeks of post DMBA injection, for histopathological and immunohistochemical and for morphological studies, respectively.
Our results indicate the chemopreventive effect of fish oil (Maxepa) on DMBA-induced rat mammary carcinogenesis. Administration of fish oil further showed a prominent reduction of cell proliferation (24.34%, P = 0.001); DPCs (25%, P < 0.001) and an increased expression of p53 protein (4.636 ± 0.19, P < 0.001) in preneoplastic mammary tissue when compared to carcinogen control counterpart. Histopathological and morphological analyses were carried out as end-point biomarkers.
Our study thus provides evidence for the anticarcinogenic effect of fish oil (Maxepa) in limiting mammary preneoplasia in Sprague-Dawley rats.
26.7 ± 2.54b
Group C (DMBA + Maxepa)
20.2 ± 1.58
The present study highlights that fish oil (Maxepa) offers considerable protection against DMBA-induced mammary carcinogenesis in female Sprague-Dawley rats. Exposure to DMBA resulted in hyperplasia that was reverted almost near to normal following treatment with fish oil, as evidenced by detailed histopathological analysis, indicating the apparent chemopreventive potential of this oil. As there is a close relationship between hyperplasia and the subsequent steps leading to malignancy, the protective role of fish oil against mammary carcinogenesis may be manifested by gradual slowing of the occurrence of hyperplasia to a considerable extent and thereby delaying the appearance of tumour with the increased latency period [15, 16].
Essential fatty acids are required for the normal structure of the membranes including cell membranes, mitochondrial membranes and nuclear membranes. They are the precursors of a whole series of second messengers  thereby regulating of the formation of cytokines, and modulate the behavior of most membrane bound proteins including receptors, ion channels and ATPase .
n-3 fatty acids may be found in vegetable oils, especially canola and soybean oil, and in green leafy vegetables as α-linolenic acid (LNA) [18:3 (n-3)] and in larger amounts in fatty cold water fish as eicosapentaenoic acid (EPA) [20:5 (n-3)] or docosahexaenoic acid (DHA) [22:6 (n-3)] . n-3 PUFA predominate in chloroplasts of green plants (linolenic acid) and in phytoplanktons (EPA and DHA). Via the marine food chain, all forms of marine life become enriched with PUFA of the n-3 family .
An inhibitory role of fish oil was clearly indicated by the reduced incidence of DMBA induced mammary tumours in rats. An observation that a significant percentage of rats having less occurrence of tumours after fish oil treatment can be explained by assuming the carcinogenic effect, even if initiated, was suppressed to a considerable extent as indicated by a long latency period of tumour development . The observed beneficial effect of fish oil on tumour growth as evidenced by the reduced tumour size may represent a selective toxicity to proliferative cells.
DPCs are shown to induce by many carcinogens . DNAs may result in large deletions during DNA replication, possibly leading to the interaction or loss of DNA sequences that may be important for tumour suppression . It has been previously reported by us that DNA protein crosslinks that occurs during experimental rat hepatocarcinogenesis, was substantially reduced by chemopreventive agent vanadium . In vitro studies by Ducore further revealed the presence of DNAs in the murine leukemia cell line L1210 that were removed in the presence of theophylline . Thus fish oil-mediated reduction of DPCs clearly demonstrates the promising inhibitory effect of fish oil to an appreciable extent in mammary carcinogenesis.
Cell proliferation is a fundamental process integral to carcinogenesis. It is considered to play an important role in several steps of carcinogenic process: initiation, promotion and progression. There are multitudes of factors known to regulate cell proliferation in breast cancer. It was found by Ip et al that selenium substantially reduced cell proliferation during mammary preneoplasia in experimental animals . Again, it was also shown by Shah et al that reduction of cell proliferation of breast cancer cell line SKBR3 occurred upon treatment with retinoic acid . Our results of cell proliferation studies did show a significant reduction of proliferating preneoplastic cells in the mammary tissue upon fish oil supplementation.
The most common anti-apoptotic lesion that is detected in cancers is the inactivation of the p53 tumour-suppressor pathway. p53 is evolved in higher organisms to prevent tumour development. Several responses can be provoked by p53, including cell-cycle arrest, senescence, differentiation and apoptosis, with the option chosen being dependent on many factors that are both intrinsic and extrinsic to the cell. In most cases, induction of p53 leads to an irreversible inhibition of cell growth, most decisively by activating apoptosis . Studies by Srivastava et al have shown that there was elevation of p53 expression during the inhibition of experimental mammary carcinomas by human chorionic gonadotropin (hCG) treatment . It was also evidenced by Kim et al that chloroform extract of Caesalpinia sappan, increased the expression of p53 in head and neck cancer cells in vitro . It can be mentioned that the inhibition of mammary preneoplasia may be mediated through the elevated expression of p53 gene. The decrease in the expression of p53 shows a parallel decrease in C-Myc and IL-Iβ-converting enzyme. Maxepa treatment showed an elevation in the expression of p53 in parallel with C-Myc and ICE genes when compared to carcinogen control group. This elevated expression was maintained for at least 15 days. This increase is also followed by significant elevations of C-Myc and p53 mRNAs. Though p53 is the most common mutated genes in human cancers, development of malignancies retain the wild-type p53 gene that is associated with its reduced activity due to overexpression of MdM2 and other cellular factors that down-regulate its level (29). Therefore, a substantial effort is presently focused on developing p53 functions in tumours harboring repressed wild-type p53.
The involvement of fish oil (Maxepa) in suppressing neoplastic transformation of mammary tissue might be mediated through the decrease of DPCs, cell proliferation and elevation of p53 tumour suppressor protein leading to apoptosis. However, its exact molecular action in combating mammary carcinogenesis requires further study. The activation of p53 by fish oil (29) may appear therefore to be a promising effective approach for the prevention of mammary tumour development.
Animals and treatment
AIN-salt mixture (g)
The entire experiment consisted of two parts: (I) histopathological and immunohistochemical estimations (II) morphological study of mammary tumors. For part (I), a total of 80 animals were divided into four groups, denoted as A, B, C and D. For histological and immunohistochemical studies (part I) twenty animals from each group (4 rats/cage) were used. At 7 weeks of age animals of group B and C were given a single, i.v. tail vein DMBA injection at a dose of 5 mg DMBA/2 ml corn oil/kg body weight in female Sprague-Dawley rats . Since, 0.2 ml of the oil was injected; the concentration of DMBA in the corn oil was 0.5 mg DMBA/0.2 ml corn oil/100 g body weight. For the morphological study (part II), a total of 250 rats were assigned to the carcinogen control group (i.e. group B) and another 250 were assigned to the DMBA + fish oil-treated group (i.e. group C). Ten animals from each group (i.e. group B and C) were sacrificed each week starting 10 weeks (17-weeks of animal age) after the DMBA injection and continued to 35 weeks (42 weeks of animal age). The treatments for various groups were as follows:
Normal control animal at 7 weeks of age were given a single tail vein injection of 2 ml corn oil/kg body weight.
DMBA-administered animals served as the carcinogen control.
DMBA + fish oil [Maxepa, (Maxepa® as manufactured by Merck Limited, Goa, India) supplemented at a dose of 0.5 ml ≈ 90 mg eicosapentaenoic acid (EPA) and 60 mg docosahexaenoic acid (DHA)] supplemented group. Each rat was given fish oil (Maxepa) orally, 2 weeks (5 weeks of animal age) prior to DMBA injection, via an intragastric tube according to Karmali et al .
Served as the fish oil (Maxepa) control.
Fish oil supplementation was started 2 weeks (5 weeks of animal age) prior to DMBA administration and continued until the termination of the experiments. The dose was given once daily, for 24 (31 weeks of animal age) weeks for histopathological and immunohistochemical analysis and 35 (42 weeks of animal age) weeks for morphological study. Fatty acid composition in the oil was determined by gas-liquid chromatography (GLC). The samples were eluted on a capillary BD23 column (J&W Scientific) after saponification with sodium hydroxide in methanol and transmethylation of the fatty acids with boron-trifluoridfe in methanol . Food and liquid consumption were monitored daily by subtracting from what was provided anything not consumed by 08:00 hours the next day, from which, the approximate food and liquid consumptions were calculated per day per rat. Close monitoring of the individual rat with marking in the tail was made. The body weight also was recorded weekly throughout the experiment and as such, there was a continuous monitoring of food and water intakes of the individual rat during the course of experiment. The animals were palpated twice a week to check for the development of palpable mammary tumours and the time of tumour appearance was also recorded.
Histological evaluation of mammary tissue
Twenty-four (31 weeks of animal age) weeks after the carcinogen or vehicle treatment, animals from each group were randomly selected; the thoracic and abdominal inguinal mammary tissues were excised from ether-anesthetized rats, fixed in 10% formalin and processed for histological studies. The tissues were dehydrated through 70, 90 and 100% alcohol and embedded in low melting-point paraffin wax. Sections of 5 μm thickness were cut and placed serially on glass slides. The sections were deparaffinized in xylene and rehydrated through 100, 90 and 70% alcohol. Three contiguous sections were made from each mammary tissue and stained with hematoxylin and eosin for histological evaluation using light microscopy. The histological slides were coded so that the particular sample identity was unknown to the individual making the assessment.
Ten animals from each group were sacrificed each week starting 10 (17 weeks of animal age) weeks after the DMBA injection and continuing for up to 35 (42 weeks of animal age) weeks. The animals were anesthetized with ether and opened by a midline incision from the pubis to the submaxillary area. The skin was dissected to expose six pairs of mammary glands. Any gross modification of the mammary fat pad by vascularization or presence of any palpable as well as non-palpable tumours was recorded. Only well-developed adenocarcinomas, confirmed through histology according to the criteria of Young and Hallows , is reported in the Results section. Tumour size was calculated using the formula for the volume of a prolate spheroid: volume = 4/3 × 3.14 × (length/2) × (width/2) × (depth/2). The width measurement was used as the depth of the tumor . The diameter measurements were further made by using a calipers and the volume of the tumor was calculated from the diameter using a stereoscopic microscope equipped with an intraocular reticule . Mammary gland was exposed for the detection of non palpable tumors only at autopsy.
Isolation of DPCs
DNA-protein complex (DPCs) from cells was isolated with slight modifications as described by Zhitkovich and Costa  and reported previously from our laboratory . A bovine serum albumin was taken as standard blank. The DPC-coefficient was determined as a ratio of the percentage of SDS precipitable DNA in treated sample to the percentage of SDS precipitable DNA in untreated control sample. Fluorescent measurements were made using model LS45 luminescence spectrometer (Perkin Elmer, UK) with NB 360 excitation and SC 450 emission filters. For protein estimation, the isolated DPCs were nuclease-digested for 1 h at 37°C; protein was estimated according to Lowry et al. .
Cell proliferation assay by immunostaining with 5'-bromo-2-deoxyuridine (BrdU)
At 6 hours before sacrifice ten animals from each group were given i.p. injections of BrdU at a dose of 50 mg/kg body weight. Only the rats in diestrus phase were used as a standard protocol. The diestrus phase was determined by histological examination of vaginal smears. The animals were anesthetized with ether and mammary tissues were dissected out. Immunohistochemical staining was done according to Zhu et al . Briefly, tissue sections were exposed to 0.3% hydrogen peroxide in ethanol for 10 min to block endogenous peroxidase and treated with 2N HCl for 1 h and incubated with trypsin 0.1% for 20 min and then with normal goat serum for 20 min at room temperature. After tissue sections were incubated with the primary antibody at room temperature (anti-BrdU mouse monoclonal antibody, Sigma) in a humid chamber (1:125 for 2 h) and then with biotinylated anti-mouse IgG (1:200; Sigma) and then with streptavidin horse radish peroxidase which binds to biotin with washing in PBS after each incubation. Visualization was revealed by reaction with 3,3'diaminobenzidine (DAB; Sigma) and 0.04% hydrogen peroxide. All slides were counterstained with hematoxylin, rinsed, dehydrated and mounted with per mount. Dark brown stains identify the cells incorporating the BrdU-label. Approximately 200 cells were counted per field, 10 fields were examined per slide and 10 slides were examined per group. The labeling index (LI) was calculated as % of BrdU positive nuclei per total number of cells counted.
Immunostaining of p53
Immunohistochemical detection of p53 protein in cold acetone fixed, paraffin embedded mammary sections was performed by the avidin-biotin-peroxidase-complex method . Briefly 5 μM thin sections on lysine-coated slides were deparaffinized and rehydrated. For immunolabeling of p53, antigen retrieval was facilitated by heating the sections in citrate buffer (pH 6.0) for 20 min. Then Endogenous peroxidase activity was blocked with 1% H2O2 in 0.1 M Tris-Nacl (PH-7.6) for 30 mins. After incubation in 5% normal goat serum sections were then separately incubated overnight at 4°C with the primary antibody, anti-sheep p53 antibody (Sigma) at 1:200 dilution in 1% BSA. Sections were then incubated with a biotinylated secondary antibody goat antirabbit IgG (Sigma) for 30 mins at 37°C with 1:100 dilutions. This was followed by incubation with streptavidin peroxidase (1:100) for 1 hour and subsequent chromagen development with 3-amino 9 ethyl carbazole (AEC-H2O2 solution) (AEC-10 mg, N, N dimethyl formamide-2.5 ml, 0.1 N acetate buffer-47.5 ml, 3% H2O2-0.5 ml.) for 4–5 min. The sections were then counterstained with Harris hematoxylin, dehydrated and mounted and served as positive control. Negative controls were prepared following all the above-mentioned steps omitting the primary antibody. Approximately 200 cells were counted per field, 10 fields were examined per slide and 10 slides were examined per group.
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