Development Of DMBA-induced Mammary Tumors Research Articles

Abstract 4642: Effects of soy protein diet on plasma isoflavones levels in obese DMBA-induced mammary tumor rat model

Abstract Obesity epidemic in the US and world grows as the number of overweight and obese adults continues to rise. Obese women have higher mortality rates from all cancers including breast cancer. Several studies have reported the protective effects of soy intake on breast cancer development, while other studies report the adverse effects of soy protein diet on breast cancer development. Soybeans contain isoflavones genistein, daidzein and glycitein, known as isoflavonoids, which are structurally similar to mammalian estrogens, have estrogenic properties and are potential anticarcinogens. Gut flora metabolizes daidzein into equol, a non-steroidal estrogen and has cancer chemopreventive property. The objective of this study was to determine the effects of obesity and a high isoflavones soy protein diet on plasma isoflavones; genistein, daidzein, glycitein, equol and O-Desmethylangolensin (O-MDA) levels using 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumor model. Five-week-old female Zucker rats (45 obese fa/fa and 54 lean) were used. After one week of acclimation (age 42 days), rats were randomly assigned to the following diet groups; 1) Lean, casein; 2) Obese, casein; 3) Lean, soy and 4) Obese, soy. Rats had ad lib access to water and diet with either casein or soy protein isolate as source of protein. All rats were orally gavaged at age 50 days with 65 mg/kg DMBA. Rats were weighed and palpated twice weekly and sacrificed 21 weeks post-DMBA treatment. Plasma was collected to measure genistein, daidzein, glycitein, equol and O-MDA levels using liquid chromatography−tandem mass spectrometry (LC-MS/MS). At end of the experiment, 69% of the Lean casein rats developed mammary tumors compared to 50% in lean soy group (p= 0.176). In the obese group, 76% of the soy-fed rats developed mammary tumors compared to 15% (P<0.001) of obese casein- fed rats. Plasma daidzein and genistein concentration were not significantly different in lean vs obese soy-fed rats. However, O-MDA and equol concentration were significantly lower (P<0.05) in obese soy-fed rats compared to lean soy rats. These data suggests that obese rats fed soy protein diet containing high isoflavone levels promoted DMBA-induced mammary tumor development compared to a casein-based diet. Also, obese soy-fed rats exhibited significantly lower plasma equol concentrations than those observed in lean soy-fed rats and this reduction may aid in explaining soy's impaired cancer chemopreventative effects in obese breast cancer model. Citation Format: Reza Hakkak, Soheila Korourian. Effects of soy protein diet on plasma isoflavones levels in obese DMBA-induced mammary tumor rat model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4642.

Effects of Obesity and 7,12-Dimethylbenz(A) Anthracene (DMBA) Treatment on Liver Cytochrome P4501A1 and 1B1 Expression in Ovariectomized Obese Zucker Rats

Obesity is a risk factor for postmenopausal breast cancer development. We have shown that obesity increases DMBA-induced mammary tumor development in intact and ovariectomized Zucker rats. Several data suggest that DMBA requires metabolic activation to exert its carcinogenic effects. The objective of this study was to investigate the effect of obesity on hepatic expression of cytochrome P450 CYP1A1 and CYP1B1 following DMBA treatment in obese and lean ovariectomized Zucker rats. Forty day-old, ovariectomized obese (n=20) and lean (n=20) Zucker rats were placed on AIN-93 G diet and 10 days later were orally gavaged with either with sesame oil (control) or with 65 mg/kg DMBA in sesame oil. All rats were sacrificed 24 hours post-DMBA treatment. Liver microsomes were prepared, and CYP1A1 and CYP1B1 expression was measured by Western blotting using goat anti-mouse CYP1A1 and CYP1B1 antibodies. DMBA treatment significantly (p<0.001) increased expression of CYP1A1 in lean and obese ovariectomized rats compared to the control group. CYP1B1 expression was not affected by obesity or DMBA. These data suggest that DMBA can increase the expression of enzymes that are responsible for DMBA metabolism in the ovariectomized Zucker rat model.

Effects of Obesity on Serum Concentration of Methylation and Oxidative/ Nitrosative Stress Metabolites Following DMBA Treatment of Female Zucker Rats

Obesity has been an epidemic in the United States for more than two decades. Studies in humans and animals have suggested that obesity is major risk for breast cancer development. It is well established that increased oxidative stress and changes in DNA can contribute to carcinogenesis. Low molecular weight antioxidant glutathione plays exceptional role in protection of cells from oxidative damage. S-adenosylmethionine, a metabolite of amino acid methionine, is a key methyl group donor for numerous of reaction including DNA. The main objective of this experiment was to investigate the effects of obesity on serum concentration of oxidative stress markers and products of oxidative damage and methylation-reaction donor S-adenosylmethionine following 7,12-dimethylbenz(a) anthracene (DMBA) treatment. Obese and lean female Zucker rats were maintained on a regular chow diet for two weeks. All rats were orally gavaged at age 50 days with 65 mg/kg DMBA. Twenty four hours after DMBA treatment, all rats were sacrificed and serum was collected. Highly sensitive HPLC method with CoulArray electrochemical detector was utilized for determination of thiols, 3-NT and SAM, SAH, and Adenosine. The obese rats had a 40% increased level of oxidized glutathione (P=0.007), decreased GSH/GSSG ratio (P=0.003), increased SAH (P=0.025) and also a 30% increased level of Ado (P=0.005) compared to lean rats. The lean animals showed a slightly higher (20%) concentration of SAM and 50% higher SAM: SAH ratio (P=0.02) compared to obese animals. These results showed that by using DMBA-induced mammary tumor development obesity can contribute to a significant imbalance in oxidative/reduction homeostasis in serum and depression in serum SAM: SAH known as methylation ratio, and increase SAH level, known as potent inhibitor methylases. These data suggest that changes plasma concentrations of glutathione, SAH and SAM reflect intracellular changes that possibly can contribute to carcinogenesis.

Effects of high-isoflavone soy diet vs. casein protein diet and obesity on DMBA-induced mammary tumor development

Obesity and elevated serum insulin growth factor-1 (IGF-1) level are major risk factors in the development of breast cancer. We investigated the long-term effects of high-isoflavone soy intake and obesity on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumor development and on serum IGF-1 and binding protein (IGFBP-3) levels. Lean and obese female Zucker rats fed casein or high-isoflavone soy protein were orally gavaged at age 50 days with DMBA and sacrificed after 147 days. The majority of lean casein-fed rats (69%) developed mammary tumors compared to 50% in lean soy-fed rats (P=0.176). In the obese groups, 76% of soy-fed rats developed mammary tumors compared to 15% of obese casein-fed rats (P<0.001). At age 43 days, IGFBP-3 was increased in the lean soy-fed rats compared to the lean casein-fed rats (P<0.05). At age 99 days, soy- and obese casein-fed rats exhibited increased serum IGF-1 compared to the lean rats and this increase was maintained for the rest of the experiment (P<0.05). Obese rats fed casein exhibited increased IGFBP-3 levels (P<0.001). However, obese rats fed soy exhibited a significant decrease in IGFBP-3 levels compared to the lean soy-fed rats (P<0.001) and a significant decrease in IGFBP-3 levels compared to the obese casein-fed rats (P<0.001). At age 197 days, IGFBP-3 levels were increased in obese casein-and soy-fed rats (P<0.001). The results suggest that female Zucker rats fed casein diets are protected against DMBA-induced mammary tumors, which is not the case for those on high-isoflavone soy diet, and changes in the concentration of serum IGFBP-3 may contribute to the incidence of DMBA-induced mammary tumors.

Dehydroepiandrosterone intake protects against 7,12-dimethylbenz(a)anthracene-induced mammary tumor development in the obese Zucker rat model

Obesity has been epidemic in the US for over two decades; almost 65% of adults in the US are overweight. Obesity has been linked with the risk of development of various cancers, including breast cancer. Dehydroepiandrosterone (DHEA) is an over-the-counter dietary supplement used as an immunomodulating, anti-depressant, anti-aging, anti-cardiovascular disease, and anti-cancer agent and anti-obesity supplement. The objectives of this study were to investigate the long-term effects of obesity and DHEA treatment on body weight gain and on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumor development. Forty-three six-week-old obese female Zucker rats were used. Rats were randomly assigned and had ad libitum access to water and a diet of either chow (2016) as a control diet or chow with the addition of DHEA at a concentration of 6 g/kg of chow as a DHEA diet. All rats were orally gavaged at age 50 days with 65 mg DMBA/kg body weight. Rats were weighed and palpated twice weekly for detection of mammary tumors and sacrificed 155 days post-DMBA treatment. Obese rats fed the DHEA diet gained significantly less weight than obese control diet rats (P<0.001). At the end of the experiment, 55% of the control diet group developed mammary tumors, while no tumors were detected in the DHEA diet group (P<0.001). Our results suggest that DHEA treatment can reduce body weight gain and protects against DMBA-induced mammary tumor development in the obese Zucker rat model.

Effects of soy with high isoflavones vs casein protein diet and obesity on plasma isoflavones levels using obese DMBA‐induced mammary tumor Zucker rat model

We reported that intact obese Zucker female rats fed semi‐purified diets were protected against DMBA‐induced mammary tumor on the casein but not soy diet. We investigate the effects of long‐term soy vs casein protein intake and obesity on plasma isoflavones levels. Five‐week‐old female Zucker rats were used. After one week of acclimation (age 42 days), rats were randomly assigned to; 1) Lean, casein; 2) Obese, casein; 3) Lean, soy and 4) Obese, soy. Rats had ad lib access to water and diet with either casein or soy protein isolate. Rats were orally gavaged at age 50 days with 65 mg/kg DMBA and sacrificed 155 days later. Plasma were collected to measure genistein, daidzein, glycitein, equol and O‐Desmethylangolensin (O‐MDA) (n=5/group). At end of the experiment, 69% of the Lean casein rats developed mammary tumors compared to 50% in lean soy group (p= 0.176). In the obese group, 76% of the soy‐fed rats developed mammary tumors compared to 15% (P&lt;0.001) of obese casein‐ fed rats. Plasma daidzein and genistein concentration were not significantly different in lean vs obese soy‐fed rats. However, O‐MDA and equol concentration were significantly lower in obese soy‐fed rats compared to lean soy rats. These data suggests that obese and lean Zucker rats have different intestinal flora which may impact DMBA‐induced mammary tumor development. (Supported by USDA and ABI to RH)

Effects of Obesity, short‐term soy vs casein protein diet and DMBA on Liver CYP 1A1 and CYP 1B1 Expression in Ovariectomized Obese Zucker Rats

Obesity is a risk factor for post‐menopausal breast cancer development. We have shown that obesity increases DMBA‐induced mammary tumor development in intact and ovariectomized Zucker rats. Several data suggest that DMBA requires metabolic activation to exert its carcinogenic effects. The objective of this study was to investigate the effect of obesity and high‐isoflavone soy (high‐isof soy) vs casein diet on hepatic expression of CYP1A1 and CYP1B1 following DMBA treatment in obese and lean ovariectomized Zucker rats. Forty day‐old, ovariectomized obese (n=40) and lean (n=40), Zucker rats were administered a diet containing either casein or high‐isof soy protein isolate and 10 days later were orally gavaged with either with sesame oil (control) or with 65 mg/kg DMBA in sesame oil. All rats were sacrificed 24 hrs post‐DMBA treatment. Liver microsomes were prepared and CYP1A1 and CYP 1B1 expression was measured by Western blotting using goat anti‐mouse CYP1A1 and CYP1B1 antibodies. DMBA treatment significantly (p&lt;0.001) increased expression of CYP1A1 in lean and obese ovariectomized rats in both casein and high‐isof soy‐fed group compared to the oil control groups. High‐isof soy diet decreased (p&lt;0.001) CYP1A1 expression in DMBA treated lean rats compared to lean casein‐fed rats but CYP1A1 expression was increased (p&lt;0.001) by DMBA in obese soy‐fed rats compared to high‐isof soy‐fed lean rats. CYP1B1 expression was not affected by diet, obesity or DMBA. These data suggest that obesity can increase the expression of enzymes that are responsible for DMBA metabolism in the ovariectomized Zucker rat model. (Supported by Susan G. Komen Foundation and ABI to RH).

Effects of Obesity on Serum Insulin‐Like Growth Factor 1 (IGF‐1) Levels Using DMBA‐Induced Ovariectomized Zucker Rat Model.

Obesity is associated with increased risk for post‐menopausal women. Several studies have suggested that IGF‐1 plays an important role in promotion and progression of breast cancer. The main objective of this study was to use DMBA‐induced ovariectomized obese zucker rat model to investigate the effects of obesity on serum IGF‐1 levels. Lean and obese rats were sham operated or ovariectomized at age 40 days and were gavaged at age 50 days with 65 mg/kg DMBA. Rats were killed 135 days post‐DMBA treatment. Serum IGF1 was measured by ELISA. Thirty‐six (36%) of the obese ovariectomized rats (O/O) developed mammary tumors while lean ovariectomized (L/O) rats developed no mammary tumors (P&lt;0.001). The obese sham (O/S) rats developed mammary tumors (59%) compared to 30% of the lean sham (L/S) rats (P&lt;0.05). Ovariectomy caused a significant (P&lt;0.001) increase in serum IGF‐1 for ovariectomized lean group but not in obese group. Also, obesity significantly increased (P&lt;0.001) serum IGF‐1 in both sham and ovariectomized rats compared to lean rats. Our results suggest that obesity is a major risk for DMBA‐induced mammary tumor development and elevated IGF‐1 levels may be associated with the increased susceptibility to DMBA‐induced mammary tumors. Other mechanisms responsible for increased mammary tumor susceptibility in obese rats are under investigation. (Supported by Susan G. Komen Foundation and ABI to RH).

1H nuclear magnetic resonance metabolomic analysis of mammary tumors from lean and obese Zucker rats exposed to 7,12-dimethylbenz[a]anthracene

Obesity increases mammary tumor development in Zucker rats following a single administration of the procarcinogen 7,12-dimenthylbenz(a)anthracene (DMBA). Fifty-day-old obese and lean female Zucker rats were orally gavaged with 65 mg/kg DMBA and sacrificed 139 days post DMBA treatment. At the end of the experiment, mammary tumors were detected in 68% of the obese rats compared to 32% of the lean group (P<0.001). 1H nuclear magnetic resonance (1H-NMR) spectra obtained for hydrophilic and lipophilic extracts from excised tumors illustrated fundamental differences in metabolic profiles between the two groups. Differences were observed for key choline compounds, namely phosphocholine and glycerophosphocholine, both markers of malignancy and apoptosis. In addition, levels of lactate, creatine, myo-inositol, alpha-glucose, alanine, leucine, glutamate, glutamine, tyrosine, phenylalanine, and NADH varied between the lean and obese groups. Principal component analysis indicated class separation between tumors from lean and obese rats based on their metabolic profiles, illustrating the potential for using 1H-NMR metabolomic methods for identifying altered metabolic pathways. Our results suggest that obesity enhances the risk for DMBA-induced mammary tumor development in rats. However, the mechanism for this increase in risk is currently unknown and will require further studies for elucidation.

Lack of a Significant Effect of Arctiin on Development of 7,12-dimethylbenz(a)anthracene-induced Mammary Tumors in Ovariectomized Sprague-Dawley Rats

: Arctiin, a plant lignan, is metabolized to hormone-like compounds with weak estrogenic and antioxidative activity in experimental animals and man. To clarify its influence on mammary carcinogenesis, female rats were administrated 7,12-dimethylbenz(a)anthracene (DMBA) once, and when the incidence of palpable mammary tumors reached 50%, subjected to ovariectomy (OVX) and divided into tumor-bearing [DMBA-Tumor (+)] and no-tumor-bearing [DMBA-Tumor (−)] groups, subgroups of each then being fed soybean-free diet containing 0, 40, 200, and 1000 ppm of arctiin for 31 wk. The incidence and multiplicity of palpable tumors in the 200 ppm DMBA-Tumor (+) subgroup from week 12 of arctiin treatment tended to be decreased as compared to the 0 ppm subgroup and at terminal sacrifice, the volume of histopathologically defined mammary tumors was decreased in the 40 ppm DMBA-Tumor (−) subgroup, but again without statistical significance. In conclusion, weak inhibitory effects of arctiin on DMBA-induced mammary tumor development were suggested in OVX rats, but any further assessment is needed to obtain conclusive results.

Isolation of two regions on rat chromosomes 5 and 18 affecting mammary cancer susceptibility

We previously mapped several quantitative trait loci (QTLs) controlling DMBA-induced mammary tumor development in female rats derived from a SPRD-Cu3 (susceptible strain) x WKY (resistant strain) cross. Two of these QTLs were assigned to chromosomes 5 and 18. In the present study, we generated and characterized congenic strains in which a segment of WKY chromosomes 5 or 18 was introduced in the SPRD-Cu3 genetic background, thereby physically demonstrating that each of these two chromosomes controls mammary tumor multiplicity. The chromosome 5 QTL (Mcstm1) accounts for 7 tumors per animal (versus a total of 11 tumors per SPRD-Cu3 rat). The chromosome 18 QTL (Mcstm2) accounts for 3 tumors per animal and is the first chemically-induced mammary cancer susceptibility locus assigned to this chromosome. In addition, the Mcstm1 region was shown to also controls tumor latency. These loci thus play a major role in chemically-induced mammary tumor development. QTLs controlling chemically-induced or estrogen-induced mammary tumor development have independently been identified on chromosomes 5 and 18, using susceptible strains others than SPRD-Cu3. Therefore the haplotype structure of the relevant chromosome regions was analyzed in the different strains. Some chromosome regions were found to be highly mosaic (haplotype blocks < 1 Mb), while one region showed an apparently conserved haplotype block of 7.5 Mb. This analysis points to limited regions that could harbor the causative genes and also indicates that at least Mcstm2 is a novel QTL.

Consumption of commercial whole and non-fat milk increases the incidence of 7,12-dimethylbenz( a)anthracene-induced mammary tumors in rats

Background: Breast cancer has become the most common cancer among women worldwide. Although the consumptions of milk and dairy products were considered to be a risk factor for breast cancer in some epidemiological studies, the results were inconsistent. Methods: In the present study, female Sprague–Dawley rats received a single oral dose of 5 mg 7,12-dimethylbenz( a)anthracene (DMBA). One week later, the animals were divided into four groups: whole milk (WM), artificial whole milk (A-WM), non-fat milk (NFM) or artificial non-fat milk (A-NFM) mixed with commercial powder chow. Rats were palpated weekly to monitor tumor development. At week 20 after DMBA administration, rats were decapitated and the volume and weight of mammary tumor were recorded. Results: Tumor incidence, the cumulative number of tumors and the sums of tumor volume were higher in the WM and NFM groups than in the A-WM and A-NFM groups both at palpation and at autopsy. Conclusion: Combining our previous studies, we found the consumption of milk promoted the development of DMBA-induced mammary tumors in rats independent of the fat level.

Promoting Effects of Milk on the Development of 7,12-dimethylbenz(a)anthracene (DMBA)-induced Mammary Tumors in Rats

To assess the effect of milk on the development of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors, 48 female Sprague-Dawley rats treated with DMBA were divided into 3 groups and given 1 of 3 test solutions for 20 weeks as their drinking liquid: milk, estrone sulfate solution or tap water. The milk group showed a significantly great incidence (75%) in tumor development compared with the water group (38%) and was comparable to the estrone sulfate group (69%). Mean tumor number per rat in the milk group was significantly higher than that in the water group (p=0.009). We classified the mammary tumors into three histological types: intraductal papilloma, fibroadenoma, and adenocarcinoma. Although the percent of intraductal papilloma and fibroadenoma was almost same among the three groups, malignant tumor was found only in the milk and estrone sulfate groups. In conclusion, our results indicate that milk as well as estrone sulfate promotes the development of DMBA-induced mammary tumors in rat and could be associated with the occurrence of adenocarcinoma.

Effects of mobile-phone microwave on dimethylbenz(a)anthracene induced mammary carcinoma development in rats

To investigate whether exposure to 900 MHz GSM wireless communication signals enhances mammary tumor development and growth induced by low dose dimethylbenz (a) anthracene (DMBA). Five hundred female Sprague Dawley (SD) rats were treated with a single dose of 35 mg/kg. DMBA and then divided into 5 groups: one control group without exposure, and 4 groups with exposure in blinded fashion. The specific absorption rates (SAR) were 0, 0.44, 1.33 and 4.00 W/kg for the 4 exposure groups, respectively. Exposure started on the next day after DMBA administration and lasted 4 hours/day, 5 days/week for 26 weeks. Rats were weighted and palpated weekly for the presence of tumors, and killed at the end of 26-week exposure period. All mammary glands were examined histopathologically. The incidence of mammary carcinoma in sham-exposure group was 37% (37/100). And mammary carcinoma incidences in the other groups of the exposure dose (0.44, 1.33 and 4.00 W/kg) were 25% (25/100), 34% (34/99) and 38% (38/100) respectively. There were no statistically significant differences between sham- and mobile phone microwave-exposed groups. In addition, the histopathological morphology of mammary tumor model in SD rats was observed. By microscopical examination two types of mammary tumor in this model were found, that was malignant or benign one. The former included adenocarcinoma and squamous cell carcinoma, and the latter included adenoma, fibroadenoma and cyst. Sometimes the histopathological morphology of mammary tumor appeared various since several kinds of histopathological features existed in the same individual. This study does not provide the evidence that 900 MHz GSM microwave exposure might promote DMBA-induced mammary tumor development in rats.

The effect of selective cyclooxygenase-2 inhibitor nimesulide on breast cancer induced by dimethylbenzoic acid in rats

To study the effect of nimesulide (NIM) on the tumorigenesis of mammary tumors induced by dimethylbenzoic acid (DMBA), and to investigate possible mechanisms of NIM against tumors. The studied rats were randomly divided into four groups: experimental control group, NIM group, diet and drug of NIM control group. The incidence of mammary tumor was observed. RT-PCR, Western blot were used to detect 8 cancerous tissues in every group, randomly. The expressions of cylooxygenase-2 (COX-2) were assessed by immunohistochemistry. The levels of prostaglandin E(2) (PGE(2)) in blood plasma and tumor tissues were determined by means of radio-immunity assay. The apoptosis index and the proliferation index were evaluated by TUNEL assay, immunohistochemical staining for proliferating cell nuclear antigen (PCNA), respectively. The incidence of mammary tumor was 69.2% in experimental control group, 40.3% in NIM group. There was obviously decreased incidence in NIM group; The expressions of COX-2 mRNA and protein were significantly down-regulated in NIM group compared with experimental control group. The increased levels of PGE(2) synthesis in blood plasma and tumor tissues were significantly decreased by administering NIM (P < 0.05). The apoptosis index was obviously higher, the proliferation index was markedly less in NIM group than experimental control group. Nimesulide could inhibit the tumorigenesis and development of DMBA-induced mammary tumors by inhibition of proliferation and induction of apoptosis. COX-2 and COX-2-mediated PGE(2) synthesis may play an important role in rat DMBA-induced breast cancer.

Effect of tamoxifen plus octreotide on DMBA-induced mammary tumors in rats

To further study the antitumor effects of TAM and somatostatin (SST) analog octreotide (OCT) in vivo. Eight weeks following dimethylbenzanthracene (DMBA, a single dose 100 mg/kg by subcutaneous injection) administration, 96 Wistar rats were randomly divided into four groups: control group, OCT (100 microg/kg bid for 14 weeks by subcutaneous injection) group, TAM (1 mg/kg 5 times weekly for 14 weeks by subcutaneous injection) group and OCT + TAM group. The mean latent phases of mammary tumorigenesis and incidence of mammary tumor-positive rats were observed. The number and volumes of tumors per animal were measured. The histological structures and ultrastructures of mammary samples were observed by using a light microscopy and a transmission electron microscopy. (1) The latent phases of mammary tumorigenesis in the TAM group and the OCT + TAM group were significantly longer than those in the control or OCT group (all P < 0.05). (2) The incidence of mammary tumor-positive rats were 70% in the control group, 52.4% in the OCT group, 45.5% in TAM group and 23.8% in the OCT + TAM group respectively, significantly longer in the three treated groups (P < 0.05 or P < 0.01), and the differences between the OCT + TAM group and the OCT group or TAM group were significant (both P < 0.05). (3) The numbers of mammary tumors per rat were markedly less in the three treated groups than in the control group (P < 0.05 or P < 0.01), and there were significantly differences between the OCT + TAM group and the OCT group or TAM group (all P < 0.05). (4) The mean volumes of mammary tumors per rat were significantly greater in the control group (6434 mm(3)) than in the three treated groups group (P < 0.05 or P < 0.01), but the tumor volume in the OCT + TAM group (1285 mm(3)) was less than those in the OCT group (4366 mm(3)) or TAM group (4138 mm(3)) (P < 0.01). At 10th week of treatment the mean volume was obviously smaller in TAM group than in OCT group (P < 0.05), but at 14th week of treatment the difference was not significant (P > 0.05). (5) Histopathological examination revealed that the mammary tumors in the OCT + TAM group were more differentiated and exhibited a less aggressive phenotype, compared with the tumors growing in the control group. Both OCT and TAM inhibit the tumorigenesis and development of DMBA-induced mammary tumors, however, resistance to TAM may appear during TAM treatment. Combination of OCT and TAM has significant synergetic antitumor effect. TAM in combination with OCT may become be an efficient hormone therapy means for breast cancer patients.

Low-fat milk promotes the development of 7,12-dimethylbenz(A)anthracene (DMBA)-induced mammary tumors in rats.

Commercial cow milk contains considerable amounts of estrogens. Our study assessed the effect of commercial low-fat milk on the development of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats. Eighty 6-week-old female Sprague-Dawley rats received a single oral dose of 5 mg DMBA. Twenty-four hours later, the animals were divided into 4 groups of 20 animals each and given 1 of 4 test solutions for 20 weeks as their drinking liquid: low-fat (1%) milk (M), artificial milk (A), estrone sulfate solution (0.1 microg/ml, E), or tap water (W). The artificial milk was formulated to supply essentially the same calories as the milk. The low-fat milk contained 378 pg/ml estrone sulfate. Tumor incidence, the cumulative number of tumors and the sum of tumor diameters were higher in the M and E groups than in the A or W groups. Overall, the development of mammary tumors was in the order: M = E > A = W. Whereas the plasma 17beta-estradiol concentration in the M group was the 2nd highest after the E group, the plasma level of insulin-like growth factor (IGF-I) was significantly higher in the M group than in the other 3 groups. In conclusion, commercially available low-fat milk promotes the development of DMBA-induced mammary tumors in rats. The degree of the promotion is almost comparable to that of 0.1 microg/ml estrone sulfate. The high estrogen content in the milk may be responsible for the promotional effects, acting in concert with other hormones such as IGF-I.

Effects of gamma-linolenic and dihomo-gamma-linolenic acids on 7,12-dimethylbenz(α)anthracene-induced mammary tumors in rats

The effects of pure gamma-linolenic acid (GLA; C18:3, n-6) and dihomo-gamma-linolenic acid (DGLA; C20:3, n-6) were investigated in 7,12-dimethylbenz(α)anthracene (DMBA) (10 mg/rat)-induced mammary tumors in Sprague-Dawley rats. 0.15 g of GLA, DGLA, or corn oil (CO) were administered (two times per week) by oral intubation, for 12 weeks to rats maintained on a 5% (w/w) CO diet. Tumor incidence, tumor multiplicity, and percent of tumor-bearing rats were highest in the CO group. Tumor multiplicity was significantly reduced in the GLA group ( p = 0.015). Feeding of GLA and DGLA resulted in significant alterations in levels of these fatty acids in phospholipids of mammary tissue, thymus, colon, liver, stomach, and ovary. These results suggest that GLA may have a small, but significant, inhibitory effect on the development of DMBA-induced mammary tumors in rats.

Prevention by dehydroepiandrosterone of the development of mammary carcinoma induced by 7,12-dimethylbenz(a)anthracene (DMBA) in the rat.

The concentration of serum dehydroepiandrosterone sulfate (DHEA-S) and DHEA decreases markedly during aging, and low circulating levels of DHEA have been associated with a higher incidence of breast cancer in women. Using 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat as model, we have studied the effect of increasing serum levels of DHEA released from Silastic implants on the incidence of these tumors in the rat. Treatment with increasing doses of DHEA leading to serum DHEA levels comparable to those observed in normal adult women (7.1 +/- 0.6 nM and 17.5 +/- 1.1 nM) caused a progressive inhibition of tumor development from 68% bearing tumors in control animals to 22% and 11%, respectively. The average tumor area per rat decreased from 2.81 cm2 in intact control animals to 0.96 and 0.09 cm2 in the groups treated with the same doses of DHEA, respectively. The present data indicate that circulating levels of DHEA similar to those found in normal adult premenopausal women exert a potent inhibitory effect on the development of DMBA-induced mammary tumors in the rat, thus suggesting the possibility of a new and more physiological approach for the prevention of breast cancer in women.

Inhibition of chemically induced mammary carcinogenesis in rats by long-term exposure to butylated hydroxytoluene (BHT): interrelations among BHT concentration, carcinogen dose, and diet.

In outbred female Sprague-Dawley rats long-term exposure to dietary butylated hydroxytoluene [3,5-di-tert-butyl-4-hydroxytoluene (BHT); CAS: 128-37-0] 1 week before carcinogen administration to termination resulted in a dose-related inhibition of mammary tumorigenesis and adrenocortical nodulogenesis. In animals fed the cereal-based NIH-07 diet and receiving a low dose (5 mg/rat) of 7,12-dimethylbenz [a] anthracene [(DBMA) CAS: 57-97-6], there was a significant overall inhibitory trend in tumor incidence observed among those receiving 300, 1,000, 3,000, and 6,000 ppm BHT. Maximal inhibition was approximately 50% at the highest concentration of BHT (6,000 ppm). The inhibitory effect of BHT on mammary tumor incidence was less pronounced when BHT was administered to rats initiated with a high carcinogen dose: At 15 mg DMBA/rat maximal inhibition was only 20% at the highest concentration of BHT (6,000 ppm). In contrast, when tumor yield was assessed in terms of latency or tumor multiplicity, the inhibitory effect of BHT was more pronounced in the groups given a high dose of DMBA than in the groups given a low dose. In animals given a low dose of DMBA (5 mg) and fed 6,000 ppm BHT in the casein-based AIN-76A diet, tumor incidence was inhibited by 50% of that of the controls; in contrast, when initiation was with a high dose of DMBA (15 mg), tumor incidence was decreased by only 28% of that of the controls. In animals fed the NIH-07 diet, DMBA-induced adrenocortical nodule formation was also inhibited in a dose-dependent fashion by BHT. At 5 mg DMBA maximal inhibition was 86% of control levels (6,000 ppm BHT); at 15 mg DMBA maximal inhibition was 66% of control levels (6,000 ppm BHT). However, when BHT was incorporated into the AIN-76A diet, its inhibitory effects on adrenocortical nodulogenesis were unexpectedly feeble and unrelated to carcinogen dose: In animals initiated with 5 mg DMBA and administered 6,000 ppm BHT, nodule incidence was decreased by only 25%, whereas in animals initiated with 15 mg DMBA, nodule incidence was decreased by 30% of that of the controls. These results indicate that while chronic exposure to dietary BHT suppressed the development of DMBA-induced mammary tumors and adrenocortical nodules, the degree of suppression depended on the dose of carcinogen administered, the level of BHT in the diet, and the parameter being measured. Diet-dependent differences in BHT action were observed with regard to DMBA-induced adrenocortical nodulogenesis but not with regard to mammary tumorigenesis.