 tumor experimental system (18). Rats were grouped three per cage and fed ad libitum with the indicated modified AIN-76A purified powdered diets. The diets were changed every 2 to 3 d.
Palpable tumors started to develop by week 5, and their incidence was recorded weekly until the time of sacrifice at week 8. At week 8 after MNU injection, rats were euthanized through CO2 asphyxiation in a numerical order across groups, equally representing the experimental groups throughout the necropsy. Blood was obtained by cardiac puncture and processed for separation of serum, plasma, and red blood cells. The palpable lesions were removed from the rat mammary fat pads, measured, and cut in half. Half of each tumor was snap frozen in liquid nitrogen. The other half was fixed in 10% neutral buffered formalin, processed, and paraffin embedded. Sections were cut at 6 μm for routine H&E staining. All lesions were histologically evaluated and scored by a certified veterinary pathologist (T.C.) blinded to the treatment according to previously published criteria in this experimental system (5). All images were obtained with an Olympus BX51 microscope and a DP71 digital camera using MicroSuite Basic 2.6 imaging software.
The two abdominal inguinal mammary fat pads were carefully excised from each rat and spread onto clean 50 × 75 mm prelabeled microscope slides (Brain Research Laboratories) for whole-mount preparation. The slides were left out for 4 to 5 min to allow the mammary fat pad to tack on, and then prepared as previously described (5). The mammary fat pads were processed for routine histologic analysis as previously reported (5).
To ensure lack of toxicity from our interventions, liver enzymes were measured in selected rats per group. In addition, their livers were removed, weighed, and subjected to histologic analysis according to previously published criteria (19).
Immunohistochemistry for Ki67 and cleaved caspase-3 was done as previously described by us (20) using a monoclonal rat anti-mouse antibody for Ki67 and a rabbit polyclonal antibody for cleaved caspase-3. Labeling was detected with EnVision + horseradish peroxidase–labeled polymer, visualized with 3,3′-diaminobenzidine, and counterstained with Mayer's hematoxylin (DakoCytomation). Scoring for Ki67 was done by one of us (R.B.), blinded to the treatment, using the image analysis software in Adobe Photoshop CS3, which recorded the number of positively stained cells in three random fields (400×) per sample. Scoring for cleaved caspase-3 (too low for adequate detection by this software) was blindly done by one of us (T.C.) by counting the number of positively stained cells in three replicate 200× fields. The number of apoptotic bodies was similarly counted by T.C. in three replicate 400× fields per tumor.
For liver enzymes, serum samples (n = 10/group) were centrifuged, aliquoted, and stored at −70°C until assayed. The liver enzymes aspartate aminotransferase and alanine aminotransferase were measured using a Cobas Mira Plus automated analyzer (Roche). Free and protein-bound GSH levels were analyzed in metaphosphoric acid extracts of tissues, and red blood cells were analyzed by an enzymatic recycling method as described previously (21).
To assess food consumption, 15 randomly selected rats per group (three rats per cage) were monitored on 5 consecutive days at weeks 7 and 8 after MNU injection. Food consumption per cage was calculated by subtracting the food leftover from that added every 2 or 3 d and divided by 3 (the number of rats per cage) to reflect consumption per rat per day. Spillage was also monitored and considered to be negligible.
Plasma fatty acids were esterified and analyzed by gas chromatography equipped with flame ionization detectors (22). The identification of fatty acid methyl esters was accomplished by comparison with the relative retention time of standards. Quantification was based on internal standard calculations compared with calibration analyses with authentic standards.
χ2 and Cochran-Mantel-Haenszel tests were used to compare the experimental groups with respect to binary (presence of tumor) and count (number of tumors) outcomes, respectively. Analysis of variance was used to compare groups with respect to continuous outcomes. All P values were corrected for multiple comparisons. All analyses were done using SAS version 9.1 (SAS, Inc.).
To verify that our dietary manipulations produced the expected changes in circulating fatty acids, we measured plasma fatty acid levels in rats fed with diets containing 5% corn oil, 20% corn oil, or 17% fish oil + 3% corn oil. The results reported in Supplementary Table S2 show that the high fish oil diet significantly increased the level of circulating n-3 PUFA and the n-3 to n-6 ratio from 0.1 to 4.4, consistent with the difference in ratio observed in the experimental diets.
As can be seen in Fig. 1, 81% of 20% corn oil–fed rats developed palpable tumors by 8 weeks following MNU administration. Neither the low-fat diet (5% corn oil) nor the n-3 PUFA–rich fish oil diet significantly affected mammary carcinogenesis by week 8 after MNU, although the latter had a transient inhibitory effect at earlier time points, which was significant at week 6 (P < 0.05; Fig. 1A). Tamoxifen administration in the presence of 20% corn oil diet inhibited mammary carcinogenesis, although its effect was only significant at week 7 at the higher dose of 100 μg/kg (P = 0.01 versus 20% corn oil; Fig. 1B). The combination of the fish oil–rich diet with the lower dose of tamoxifen of 50 μg/kg significantly inhibited carcinogenesis at week 6 (P < 0.01), week 7 (P = 0.01), and marginally (P = 0.06) at week 8 (Fig. 1C). At termination, this combination had reduced tumor development by 35%, a reduction of borderline statistical significance. In contrast, tamoxifen alone at this dose did not affect mammary carcinogenesis significantly at any time points (Fig. 1C). However, the effect of the combination treatment was not found to be statistically different from that of tamoxifen alone when compared directly. Remarkably, the combination of the higher dose of tamoxifen (100 μg/kg) with fish oil–rich diet profoundly inhibited mammary carcinogenesis at week 6 (P < 0.001), week 7 (P < 0.001), and week 8 (P = 0.002; Fig. 1D). Overall, this combination reduced tumor formation by 47% by the end of the experiment. In contrast, the inhibitory effect of tamoxifen at this dose on mammary carcinogenesis was only significant at week 7 (P = 0.01; Fig. 1D). However, direct comparison of the effects of the combination with that of tamoxifen alone was not statistically different.
Effects of dietary interventions and tamoxifen administration on mammary carcinogenesis. A-D, groups of 21-d-old MNU-injected rats (n = 35-39 per group) were randomly assigned to the indicated experimental interventions (see Materials and Methods). See text for statistical analysis. CO, corn oil; FO, fish oil; TAM50, 50 μg/kg tamoxifen, s.c., 5 d/wk; TAM100, 100 μg/kg tamoxifen, s.c., 5 d/wk.
Table 1 illustrates the effects of our intervention on the multiplicity and volumes of the palpable lesions. Neither the low-fat diet nor the fish oil–rich diet significantly affected tumor multiplicity. Starting at week 6, the combination of fish oil–rich diet and 50 μg/kg tamoxifen significantly reduced tumor multiplicity (group 6), whereas the individual interventions (groups 3 and 5) did not. Increasing the dose of tamoxifen to 100 μg/kg reduced tumor multiplicity (group 4). However, the combination of this dose of the drug with a fish oil–rich diet (group 7) exerted a more significant suppressive effect. Direct comparison of the effects of each combination with those of each dose of tamoxifen alone (50 or 100 μg/kg) did not reveal statistically significant differences. The combination of fish oil–rich diet with 100 μg/kg tamoxifen was the only intervention able to cause a significant reduction in the tumor volume, whereas the effect of 50 μg/kg tamoxifen plus fish oil–rich diet did not reach statistical significance (P = 0.06). When non–tumor-bearing rats were excluded from the analysis, no differences in tumor volume were observed across groups (data not shown).
Supplementary Table S3 reports the histopathology of the palpable lesions. The vast majority were invasive adenocarcinoma, 14% of which with an associated ductal carcinoma in situ (DCIS) component. Next, we analyzed the effects of our treatments on the incidence and multiplicity of invasive adenocarcinomas (with and without DCIS) subdivided according to histologic subtypes (Table 2). The low-fat diet (5% corn oil) and the fish oil–rich diet by themselves did not affect tumor incidence or multiplicity. The combination of the fish oil–rich diet and 50 μg/kg tamoxifen significantly reduced total tumor multiplicity by ∼50% (group 6), whereas the individual interventions did not (groups 3 and 5). Administration of 100 μg/kg tamoxifen to corn oil–fed rats significantly (P < 0.05) reduced multiplicity of papillary and total number of adenocarcinomas (group 4). However, the combination of 100 μg/kg tamoxifen with the fish oil–rich diet (group 7) exerted a more marked effect, reducing the incidence of cribriform tumors by 52% (P < 0.05); the incidence and multiplicity of papillary tumors by 73% and 83%, respectively (P < 0.01 for both); and the incidence and multiplicity of all tumors combined by 47% and 65% (P < 0.01 and P < 0.