High maternal milk intake in the postnatal life reduces the incidence of breast cancer during adulthood in rats

Environmental factors during perinatal life can lead to changes in the mammary gland, making it susceptible to cancer in adulthood. Breastfeeding has a special importance since it takes place at a critical period of growth and development of the newborn. We aimed to analyze if an appropriate lactation protects the offspring against mammary carcinogenesis during adult life and explore the mechanisms involved in the protective effect. One-day-old Sprague-Dawley female rats were randomly distributed in litters of three (L3), eight (L8) or 12 (L12) pups per dam, to induce a differential consumption of breast milk. At 55 days of age, the animals were treated with a single dose of dimethylbenzanthracene to study tumor latency, incidence and progression. Histological, immunohistochemical and Western blot studies were performed. We observed lower incidence and higher latency in L3 compared to the other groups. The mitotic index and expression of proliferating cell nuclear antigen (PCNA) was significantly augmented in tumors of L12 rats compared to L3 and L8, while the apoptotic index was augmented in tumors of L3 v. L12. Cleaved caspase 8 was significantly higher in tumors from L3 compared to L12. Tumors developed in L3 have a greater number of apoptotic bodies and a greater expression of caspase 8. These results demonstrate that the animals that maintained a higher intake of maternal milk (L3) presented lower incidence and greater tumor latency. Lower consumption of breast milk (L12) would increase tumor mitosis and the expression of PCNA, explaining the higher tumor incidence observed in this group.

Métodos

Postnatal litter size adjustment Female Sprague Dawley rats bred in our laboratory were used. The animals were kept in a light (lights on 06.00–20.00 h) and temperature (22–24°C) controlled room. One day-old female pups (n = 51) born on the same day were distributed ad random in litters of different sizes: three (L3), eight (L8) or twelve (L12) pups per dam, to induce a differential consumption of maternal milk. The size of the litters was chosen in accordance with previous studies16,17. It has been previously demonstrated that fostering of one-day old pups of the Sprague Dawley strain does not lead to any adverse effect18. Body weight of pups was monitored every 3 days. At day 21, the litters were weaned and fed with rat chow (Cargill, Argentina) and tap water ad libitum until the end of the experiment. They were housed in cages containing approximately 6 rats from the same group per cage. Body weight was measured once a week in order to elaborate their growth curves. In this way we obtained three groups of rats growth with different levels of lactation: L3 (n=14), L8 (n=23) and L12 (n=14). Induction of mammary tumors At the age of 55 days all rats were treated per os with a single dose (15 mg/rat) of 7,12-dimethylbenzanthracene (DMBA, Sigma-Aldrich, USA) by an intragastric probe, three hours after food and water deprivation to ensure a complete absorption of the drug. DMBA has been extensively used at that dose to study mammary carcinogenesis19,20,21. The animals were palpated twice a week. Latency, incidence and progression of tumors were determined in all groups. Latency, incidence and progression of tumors The latency was considered as the time between DMBA administration, and the appearance of the first palpable tumor. Incidence was calculated as the percentage of rats that had tumors within the study period respect to the total number of rats per group. We used a caliper to measure the major (DM) and minor (dm) diameters of the tumors twice a week and calculated the tumor volume (TV) as TV = dm2 x DM/2. Tumor progression was assessed estimating tumor growth rate (GR) as GR = TV/(day of sacrifice-day of appearance of first tumor). Sample collection All the animals were decapitated between 10:00 and 12:00 h on the day of diestrus when the tumors reached a volume ≥ 1,000 mm3 or at the end of the experiment on day 250 if they did not develop any mammary tumor22. When more than one tumor developed in a single rat, the volume limit of 1,000 mm3 was considered for the first tumor reaching that value. Immediately after decapitation, intra-abdominal fat was removed, weighed and expressed as a percentage of total body weight. A piece of each tumor was removed for histopathological and immunohistochemical (IHC) analysis. Tumor histology Small pieces of each tumor were processed for histopathologic studies by fixing in buffered formalin, dehydrating in ethanol and embedding in paraffin wax. Sections (3–5 μm) were cut with a Hyrax M 25 microtome and stained with hematoxylin and eosin (H&E) to classify tumors according to published criteria23,24. Images were captured with an Eclipse E200 microscope coupled to a CCD camera with 5.0M resolution, and were analyzed with Micrometrics SE Premium (both from Nikon Corp., Japan) under magnification of 100x, 400x and 600x. Apoptotic and mitotic indexes Ten fields of histological sections of each tumor (6-19 tumors/group) stained with H&E were observed with a magnification of 400x. The counting of mitotic and apoptotic figures was performed in double-blind by two independent observers, avoiding fields with necrosis, inflammation or tissue folds. Mitotic and apoptotic indexes were calculated from this count. The mitosis/apoptosis ratio (M/A ratio) was calculated by dividing the mitotic index by the apoptotic index from each tumor. Immunohistochemistry Serial sections (3–5 μm) were mounted onto 3-aminopropyltriethoxysilane (Sigma-Aldrich, Argentina)-coated slides for subsequent IHC analysis. An antigen retrieval protocol using heat to unmask the antigens was used (30 min in citrate buffer, 0.01 M, pH 6.0). Tissue sections of 6-19 tumors/group were incubated overnight at 4°C in humidity chambers with the primary antibodies anti-proliferating cell nuclear antigen (PCNA, Dako Cytomation, Denmark) and anti-Ki67 (Abcam, USA). A commercial kit to detect mouse antibody was used (Dako EnVision system, horseradish peroxidase, diaminobenzidine; Dako, USA). Slides were lightly counterstained with hematoxylin to reveal the nuclei, examined and photographed. The percentage of positive nuclei was obtained based on an average of 700 cells counted per sample, at 400x magnification. We used a scoring system reported previously25. Briefly, we used an intensity score 0 = no staining, 1 = nuclear staining of <10% of tumor cells, 2 = staining between 11 and 33% of tumor cells, 3 = staining between 34 and 65% of tumor cells, 4 = staining of >66% of tumor cells. These scores were obtained by two independent observers blinded regarding the experimental group, and a few conflicting scores were resolved by consensus. Western blotting Total proteins were extracted from tumors and western blot was performed as described previously22,23. Fifty μg of protein were separated by SDS–PAGE and electrotransferred to Immun-Blot® PVDF Membranes (BIO-RAD, USA) overnight at 4°C. After rinsing and blocking with BSA 0.5% the membranes were incubated overnight with corresponding primary antibodies anti-Bcl-2 (Santa Cruz Biotechnology Inc, USA); anti-Caspase 8 and anti-Bax (Abcam, USA); anti-Cleaved Caspase 9 (Cell Signaling Technology Inc, USA); BID (Santa Cruz Biotechnology Inc, USA) and then, with the corresponding horseradish peroxidase-conjugated secondary antibody goat anti-rabbit or goat anti mouse (Santa Cruz Biotechnology Inc, USA). Protein expression was detected by enhanced chemiluminescence (Amersham ECL™, GE Healthcare, Argentina) using a ChemiDoc XRS+ System with Image Lab Software from BIO-RAD to detect specific bands that were quantified by densitometry using FIJI Image processing package26. The membranes were stained with ß-Actin (Santa Cruz Biotechnology Inc, USA) as loading and transfer control. Estimation of milk intake in pups from different sizes of litters The average increase in weight of the litter during a lactation period serves as an estimate of the amount of milk secreted during the secretion interval. To determinate this, litters of 3, 8, or 12 pups per dam (n = 5 mothers in each group), were separated from their mothers at 07:00 h on days 10, 11 and 12 of lactation, during 4 h (separation interval). Before being returned to the mothers, the pups were weighed individually. Then they were allowed to suck for 60 minutes. At the end of that suckling time, the pups were weighed again. The results were expressed as the average of the individual weight increases per litter27. Data from days 10 and 11 were discarded assuming that dams and pups were adapting to the disruption of their normal suckling routine during that period, to minimize any stress during the experiment on day 12 when the data were actually recorded. Triglycerides, proteins and lactose in milk Milk was obtained on day 14 of lactation from five mothers per group with litters of 3, 8, or 12 pups per dam. Pups were separated from their mothers at 07:00 h; 4 hours later, the dams were lightly anesthetized with ketamine (1 ml/kg) and xylazine (0.5 ml/kg), administered with an intraperitoneal injection of 1 IU of oxytocin, and milk was extracted by gentle pressing the nipples as previously described28. About 0.5 ml of milk was obtained from each rat and kept frozen in microvials until determinations. Concentrations of triglycerides (TGs), protein and lactose were determined by the end-point colorimetric method, in a Roche's clinical chemistry analyzer Cobas c311.

Palabras clave: CASPASE 8, DMBA, MAMMARY GLAND, PCNA

Alcance geográfico

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Identificador del recurso

URI: http://hdl.handle.net/11336/190516

Colecciones

Datos de Investigación(IMBECU)
Datos de Investigación de INST. DE MEDICINA Y BIO. EXP. DE CUYO

Citación

Santiano, Flavia Eliana; Pistone Creydt, Virginia; López Fontana, Constanza Matilde; Caron, Ruben Walter; (2023): High maternal milk intake in the postnatal life reduces the incidence of breast cancer during adulthood in rats. Consejo Nacional de Investigaciones Científicas y Técnicas. (dataset). http://hdl.handle.net/11336/190516

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