Antiestrogen-binding sites in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors: relation to growth

We have detected specific high-affinity binding sites for nonsteroidal antiestrogens in 98% of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. Since recent studies have suggested that these binding sites may be involved in the regulation of cell growth and proliferation, we attempted to define a possible relationship between the growth of these hormone-dependent tumors and their antiestrogen-binding site content. Rats bearing such tumors were either treated with haloperidol (to increase prolactin secretion and stimulate tumor growth) or oophorectomized (to reduce circulating estrogen concentration and suppress tumor growth). Compared with controls, haloperidol treatment clearly enhanced tumor growth while oophorectomy induced tumor regression, but neither procedure had any effect on the antiestrogen-binding site concentration. Furthermore, tumors which responded to endocrine manipulation had similar antiestrogen-binding site concentrations as tumors which did not respond. We conclude that (1) the alterations in tumor growth induced by these endocrine manipulations are probably not mediated through a change in antiestrogen-binding site concentration, and (2) the tumor concentration of these binding sites is not under estrogen or prolactin control.     

The effects of high dietary fat and indomethacin on 7,12-dimethylbenz(a)anthracene-induced mammary carcinomas in rats

The effects of high dietary fat and indomethacin (IND) on tumorigenesis, tumor growth, tumor histology, cell kinetics and receptor contents of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinomas were investigated in Sprague-Dawley (S-D) rats. Tumorigenesis was stimulated by high dietary fat and inhibited by IND. However, both high dietary fat and IND stimulated tumor growth. Switching the animals from the high-fat diet with or without IND to the low-fat diet suppressed tumor growth and affected the cell kinetics. Histologically, the tumors showed a dense proliferation of ductal cells in the high-fat alone or IND-treated high-fat diet group. Tumors in the low-fat or IND-treated low-fat group, as well as the group switched from a high-fat or IND-treated high-fat diet to a low-fat diet, demonstrated well-developed glandular structures. The estrogen receptor and progesterone receptor contents were not different between the groups either prior to or following switching diets. Therefore, high dietary fat stimulates tumorigenesis and tumor proliferation, while IND has dual effects: a stimulating effect on tumor proliferation, but an inhibiting effect on tumorigenesis. It also appears that hormone-receptor status does not play an important role in the stimulatory effects of high-fat diet or IND on tumor proliferation.     

Comparative effects of a series of prolactin inhibitors, 17beta-estradiol and 2alpha-methyldihydrotestosterone propionate, on growth of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinomas.

Eight ergot alkaloids and ergoline derivatives, effective prolactin inhibitors, were tested for activity against DMBA-induced rat mammary carcinomas. Compounds were administered daily, 5 times/week for 4 weeks, and rats were observed for an additional 4 weeks. Groups treated with androgen and estrogen were used as positive controls. Those ergot compounds and ergolines that proved to be highly effective in reducing tumor size or in inducing regression of tumors to nonpalpability were Deprenon (D-6-methyl-8-ergolin-I-ylacetic acid amide) and ergocryptine; effective to an intermediate degree were Dironyl [N-(D-6-methyl-8-isoergolin-I-yl)-N',N'-diethylurea], ergocornine, and Lysenyl [N-(D-6-methyl-8-isoergolenyl)-N',N'-diethyl-urea]; and effective to a minimal degree were Lergotrile (2-chloro-6-methylergoline-8beta-acetonitrile), CB-154, and 6605-VUFB (D-6-methyl-8-cyanomethylergolin-I). Remission of many individual carcinomas was brief, and duration of complete regression (all tumors in the rat were nonpalpable) was less than 10 weeks.     

Therapy of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinomas with combinations of 5-fluorouracil and 2 alpha-methyldihydrotestosterone propionate

This investigation was undertaken to determine whether a combination of a cytotoxic drug with a sex hormone would provide efficacious therapy for mammary carcinomas. Established, 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinomas were treated with 5-fluorouracil (5-FUra) and 2 alpha-methyldihydrotestosterone propionate (MDTP) for 4 weeks. At end of therapy, pooled data showed 21% of the tumors in complete remission (CR) in rats given 5-FUra at 17.5 mg/kg/day and 3% in those given 8 mg/kg/day. Administration of MDTP at 1.25 to 5 mg/kg/day yielded 15 to 48% tumor CR. The combination of 5-FUra at 17.5 mg/kg/day with MDTP at 5, 2.5, and 1.25 mg/kg/day induced, respectively, 96, 91, and 75% CR. Maxima of 100, 100, and 92% CR were obtained in single tests at these respective doses. Therapy with combinations of 5-FUra at 8 mg/day and MDTP at 2.5 and 1.25 mg/kg/day yielded, respectively, 69 and 61% tumor CR. Appearance of new tumors during and after therapy was controlled more effectively by combinations of the two agents. Analysis of percentage of tumor CR showed marked synergism for 5-FUra and MDTP. A second course of combination therapy effectively prolonged duration of CR. Therapy with the cytotoxic drug 5-FUra in combination with the androgen analog MDTP is highly efficacious against induced mammary carcinomas.     

Vascular reactivity and perfusion characteristics in 7,12-dimethylbenz(a)anthracene-induced rat mammary neoplasia

Blood flow during "resting conditions" and during noradrenaline infusion were studied by the labeled microsphere technique in dimethylbenz(a)anthracene-induced mammary tumors, skin, muscle, and lung in the rat. Intratumoral distribution of flow was studied by autoradiography of spheres trapped in the vascular beds of the tumors. Histological examination was performed and correlated to the blood flow data. Mean blood flow to the tumors during "resting conditions" was relatively high (49 ml/min/100 g tissue) but was substantially decreased (5 ml/min/100 g tissue) during noradrenaline infusion which produced a pressure elevation of 35 mm Hg. Thus, vascular resistance of the tumor tissue increased dramatically. Cardiac output increased, but total systemic resistance was unchanged. Vascular resistance in muscle was unchanged in contrast to an increase seen in skin. Shunted systemic blood flow to the lungs and bronchial arterial flow decreased indicating reactivity of abnormally large arteriovenous passages in the tumors. Poorly differentiated tumors had a higher vascular resistance than did well-differentiated tumors. Autoradiography revealed a nodular flow distribution with a slight tendency of higher perfusion in the periphery of these tumors.     

Relationship between insulin and estrogen binding to growth response in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors.

Insulin and estrogen binding have been determined in 7,12-dimethylbenz(a)anthracene-induced mammary tumors of rats in various endocrine states. Hormonal therapy, such as diabetes and ovariectomy, resulted in differential effects on growth patterns and hormone binding of tumors coexisting in the same host or in different hosts. It was observed that tumors that continued to grow after the host was made diabetic (insulin independent) or started to regress after ovariectomy (ovarian dependent) demonstrated decreased insulin binding. Tumors that regressed in diabetic hosts (insulin dependent) or continued to grow in ovariectomized animals (ovarian independent) showed an increased insulin-binding capacity. No significant change in insulin binding was observed in tumors that remained static after ovariectomy or induction of diabetes. Estrogen binding in tumor cells from diabetic rats paralleled the pattern of tumor growth response to diabetes; insulin-independent tumors demonstrated a significant increase in binding compared to tumors from intact hosts, and insulin-dependent tumors showed decreased estrogen receptor levels. From these results, we conclude that (a) insulin plays a positive role in regulating estrogen-binding capacity, (b) ovarian hormones may play a role in regulating insulin-binding capacity, and (c) a relationship between insulin and ovarian hormones and the growth of 7,12-dimethylbenz(a)anthracene-induced tumors is strongly suggested and may have therapeutic implications.     

Age as a modifying factor of 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis in the lewis rat.

The influence of age at DMBA administration on (1) susceptibility to mammary tumor development, (2) the proportion of tumors which are ovary-dependent, and (3) the frequency of spontaneous tumor regression was examined in female Lewis rats between 25 and 200 days of age. Tumor development was monitored for a 12-month period after carcinogen administration. Age at carcinogen administration affected (1) the number of tumors induced, (2) the latent period of tumor development, and (3) the number of tumors which regressed spontaneously. More tumors developed in young rats; however, a large fraction of these tumors regressed spontaneously. The frequency of spontaneous tumor regression was strongly age-related, being highest in rats treated up to 70 or after 150 days of age. Mean latent period of development of tumors which regressed spontaneously (SRT) was significantly shorter than that of tumors which grew progressively (PGT). Furthermore, mean latent period of SRT development was significantly shorter in rats treated at 25 or 50 days of age. In contrast, all age-groups were equally susceptible to the development of progressively growing tumors (PGT). PGT incidence, mean latent period of PGT development, mean numbers of PGT per PGT-bearing rat and the relative proportion of PGT which were ovary-dependent were similar for all age-groups at the end of 12 months. These results indicate that, in the Lewis rat strain, age does not confer significant or permanent refractoriness. Furthermore, spontaneous tumor regression is a frequent and strongly age-related phenomenon.     

Possible enhancing effects of atrazine on growth of 7,12-dimethylbenz(a) anthracene-induced mammary tumors in ovariectomized Sprague-Dawley rats

Atrazine, one of the most commonly used herbicides in the world, has been reported to have endocrine disrupting effects in vivo. In the present experiment, influence of dietary atrazine on the late promotion/progression stage of mammary carcinogenesis in ovariectomized female Sprague-Dawley rats was examined after a single intragastric administration of 7,12-dimethylbenz(a)anthracene (DMBA). When the incidence of palpable mammary tumors reached about 50%, the animals were subjected to ovariectomy and divided into tumor bearing [DMBA-Tumor(+)] and non-tumor bearing [DMBA-Tumor(-)] groups, with subgroups of each fed a soybean-free diet containing 0, 5, 50, or 500 p.p.m. atrazine for 34 weeks. At the completion of the study, the tumor volume in the 50 and 500 p.p.m. treatment Tumor(+) subgroups was greater than in the 0 p.p.m. control case. In the DMBA-Tumor(-) group, higher incidences and volumes of the mammary tumors, with or without statistical significance (P <0.05), were observed in the 50 and 500 p.p.m. subgroups. Atrazine treatment tended to increase proportion of estrogen receptor alpha-positive tumors and stimulated cell proliferation in the DMBA-Tumor(+) group, but with no clear effects on serum hormone levels. The present study indicates that atrazine has a potential for enhancing the growth of mammary tumors, partly through increasing cell proliferation in the promotion/progression stage in female rats under ovarian hormone-free conditions.     

Effect of short-term feeding of sodium selenite on 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis in the rat

The inhibitory activity of short-term feeding of one of four concentrations of dietary selenium against the induction of mammary gland carcinomas by 7,12-dimethylbenz(a)anthracene (DMBA) was studied in female Sprague-Dawley rats. When 28 days old, the animals were placed on a Torula yeast diet formulation which contained, by analysis, either 0.05, 0.15, 1.05, or 2.06 microgram of selenium, as sodium selenite, per g of diet. Mammary cancer was induced by a single p.o. administration of either 7.5 or 15.0 mg DMBA at 50 days of age. The animals were maintained on the above diets until 14 days after carcinogen treatment at which time all animals were transferred to a chow diet containing 0.21 microgram of selenium per g of diet. The study was terminated 120 days after DMBA administration. The concentrations of selenium in the liver and mammary tissue measured at the time of DMBA treatment increased with increasing levels of dietary selenium (p less than 0.05). At the low dose of DMBA, there was a trend towards reduction in the number of cancers with increased amounts of selenium, but the only significant difference occurred between groups fed the next to lowest and the highest level of selenium. At the high dose of DMBA, the number of observed cancers showed a strong dose effect (p less than 0.05). In addition, tumor load was significantly reduced in selenium-supplemented rats (p less than 0.05), and there was a significant delay (p less than 0.05) in the time to appearance of the cancers of animals receiving the highest level of selenium when compared with those receiving the lowest level. The dietary concentrations of selenium shown to inhibit the early stage(s) of cancer induction in this system were both significantly lower and fed for a shorter time interval than that which was previously reported.     

Growth inhibition of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors by controlled-release low-dose medroxyprogesterone acetate

Summary Since our previous findings had indicated that the androgenic steroid medroxyprogesterone acetate (MPA) exerts potent inhibitory effects on 7,12-dimethylbenz(a)anthracene (DMBA)-induced tumor growth, we have studied the effect of low doses of MPA released from Depo-Provera and from 50:50 poly[DL-lactide-co-glycolide] microspheres in the same DMBA-induced tumor model. The present data show that single subcutaneous injection of a 4-month controlled-release formulation of biodegradable 50:50 poly[DL-lactide-co-glycolide] microspheres containing 10 mg of MPA giving serum levels of 3.14±0.32 ng/ml (8.12±0.83 nM) MPA causes a maximal or near-maximal 60% inhibition of tumor growth measured 56 days later. Such data suggest that controlled-release formulations giving constant and low blood levels of MPA could be used for the treatment of breast cancer in women. Such a low concentration of MPA should avoid the side effects observed with the high doses of the compound.     

Optimization of 7,12-dimethylbenz(a)anthracene-induced rat epithelial ovarian tumors

Ovarian carcinoma is the second most common malignant tumor of the female reproductive system and an notable cause of cancer death. The detection and diagnosis of early ovarian carcinomas are still clinical challenges, which calls for imaging studies using early ovarian carcinoma animal models. The present study aimed to optimize the 7,12-dimethylbenz(a)anthracene (DMBA)-induced model of rat ovarian tumors by investigating the delivery methods, induction dose and time of DMBA exposure, and explored the morphological features of tumors using MRI. Three schemes were performed. In scheme one the ovary was covered with absorbable hemostatic gauze loaded with a high concentration of liquid DMBA. For this scheme, 150 Sprague-Dawley rats were divided into three groups depending on the DMBA dose (1.0, 2.0 and 3.0 mg). In scheme two DMBA solution was injected under the ovarian capsule. For this scheme, 159 rats were divided into 0.5, 1.0 and 1.5 mg DMBA groups. In scheme three the ovary was covered with absorbable gauze loaded with a high concentration of solid DMBA. For this scheme 161 rats were divided into 1.0, 2.0 and 3.0 mg DMBA groups. Each group of the three schemes was further subdivided into 60-, 90-, 120-, 150- and 180-day groups. In scheme two, the tumor formation rate was 75.6% (99/131), which was the highest in the 1.5 mg group (86.4%, 38/44) and reached 100% (10/10) on day 120. The induced tumors were serous in 93.9% (93/99) of tumors. Borderline ovarian tumors accounted for 19.2% (19/99) of all tumors, and ovarian cancer accounted for 46.5% (46/99). The mean maximum diameter (MMD) of borderline ovarian tumors was 10.29±3.41 mm, and that of ovarian cancer was 15.19±7.10 mm. MMD of the solid components increased with increasing malignancy. Cystic, cystic-solid and solid tumors were observed. The ovarian subcapsular injection of 1.5 mg DMBA was the best scheme for the rat ovarian tumor model. The present model is ideal for investigating the occurrence, development and imaging of ovarian tumors.     

Inhibition of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinogenesis by concomitant or postcarcinogen antioxidant exposure

When administered prior to or at the time of carcinogen exposure, the phenolic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are effective inhibitors of carcinogenesis in several target organs. However, chronic, postcarcinogen administration of BHT apparently enhances tumorigenesis in certain animal models for liver and lung cancer. The present study was performed to determine the effects of BHA and BHT on mammary carcinogenesis when antioxidant exposure is limited to defined periods encompassing or following carcinogen availability. At 50 days of age (Time O), virgin female Sprague-Dawley rats (25/group) were given a single intragastric dose of 8 mg of 7,12-dimethylbenz(a) athracene . Basal diet (Wayne Lab Meal) was supplemented with 5000 or 2500 mg of BHA or BHT/kg by the following protocol: 2 weeks before until 1 week after carcinogen administration; 1 week after carcinogen administration until the end of the study; or none. The experiment was terminated 210 days after 7,12-dimethylbenz(a)anthracene administration, and all mammary tumors were confirmed histologically. When administered by the 2 weeks before to 1 week after schedule, both BHA and BHT were effective inhibitors of mammary carcinogenesis. However, the compounds also were active in chemoprevention when administered by the 1 week after to end protocol. These data indicate that the anticarcinogenic activity of antioxidants is not limited to influences on carcinogen metabolism, since both BHA and BHT inhibited mammary tumor induction when their administration was begun following clearance of the carcinogen from the mammary gland. The anticarcinogenic activity of postcarcinogen administration of BHA and BHT in the mammary gland is in contrast to the apparent tumor-enhancing activity of BHT in the liver and lung.     

Potent inhibitory effect of a new antiestrogen (RU 16117) on the growth of 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors.

At the daily dose of 24 mug for a period of 4 weeks, RU 16117 (11alpha-methoxyethinyl estradiol), a new antiestrogen, led to 65% reduction of the number of already established dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in female Sprague-Dawley rats. Not only the tumor number but also the tumor size was reduced by RU 16117 in a manner similar to that seen after ovariectomy. The absence of an inhibitory effect of doses of 0.1 to 12.5 mug 17beta-estradiol (E2) per day, a dose-range which covers the low estrogenic activity of the RU 16117 doses used, suggested that the inhibitory effect of RU 16117 was not due to its estrogenic activity. Decreased levels of receptors for E2, progesterone, and prolactin were found in the tumors remaining after ovariectomy; treatment with the dose of RU 16117 sufficient to inhibit tumor growth (24 mug) had a similar inhibitory effect on the levels of E2 and prolactin receptors. These data suggested that a reduction of hormone receptor levels in the tumor tissue could be a mechanism by which RU 16117 acts as a potent inhibitor of the growth of DMBA-induced mammary carcinoma.     

Modification of the effect of a gonadoliberin analog on 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors by hormone replacement.

A gonadoliberin analog, (D-leucyl6, desglycyl-NH2(10), prolyl ethylamide9) gonadoliberin, is known to suppress ovarian function and plasma prolactin levels. Its antitumor activity was evaluated against mammary tumors induced in Sprague-Dawley rats by dimethylbenz(a)anthracene. Observations were made when the analog, referred to as A-43818, was given alone and together with estrogen replacement or perphenazine, A-43818, 10 microgram s.c. twice a day for 6 weeks, was highly effective in producing tumor remissions. All of the 11 animals survived throughout the observation period, complete regressions occurred in 8 of 13 tumors, and 2 were classified as static. None of the 16 tumors in 12 control rats regressed, and there were 4 deaths. When estradiol benzoate, 2 microgram s.c. each day, was administered with the A-43818, antitumor activity was suppressed; only 2 of 17 tumors regressed, 6 were static, and 5 of the 10 rats in this group died. Perphenazine, 1 mg i.m. daily, a dose known to cause hyperprolactinemia, also impaired the efficacy of A-43818. Three of 14 tumors regressed, 6 were static, and the rest continued to grow; 3 of the 12 rats died within 6 weeks of starting treatment.     

Antagonism of development and growth of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors by the antiestrogen U 23,469 and effects on estrogen and progesterone receptors.

This study analyzed the effectiveness of a nonsteroidal antiestrogen, cis-(3-[p-(1,2,3,4-tetrahydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]-1,2-propanediol (U 23,469) previously shown to be potent in antagonizing estrogen-induced uterine growth, in preventing the development of 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors and in eliciting the regression of established tumors; the study also attempts to elucidate the mechanisms of the tumor antagonism of U 23,469. Virgin female Sprague-Dawley rats that receive DMBA at 47 to 50 days of age and then receive U 23,469 (250 micrograms s.c. in 0.15 M NaCl daily) have a greatly reduced number of mammary tumors and a markedly decreased tumor area. Treatment with U 23,469 for increasing time periods (3, 6, or 12 weeks) beginning 2 weeks after DMBA results in a progressive delay in onset of tumor appearance. U 23,469 treatment beginning 1 week after DMBA or given prior to DMBA is even more effective. The time course of tumor regression (3 months after DMBA) by U 23,469 or ovariectomy is similar, with 50% regression in ca. 2 weeks, and both elicit regression of almost all tumors (greater than 90%). After ovariectomy, cytosol progesterone receptor levels are greatly diminished in tumors and uteri, while cytosol estrogen receptor (ER) levels are high; in both tissues little (ca. one-third) of ER is in the nucleus. During U 23,469 treatment, cytosol ER content is very low in regressing tumor and uterus and over 90% of ER is in the nucleus; cytosol progesterone receptor is slightly depressed in the uterus but is at the untreated level in mammary tumor. These receptor studies suggest that the effectiveness of this antiestrogen in antagonizing mammary tumor development and growth may reside in its ability markedly to perturb the distribution of ER, maintaining over 90% of ER in the nucleus with concomitant low levels of cytoplasmic ER, a situation that may render the mammary tissue insensitive to the animal's own endogenous estrogens.     

Estrogen-prolactin dependency in 7,12-dimethylbenz(a)anthracene-induced tumors.

Hormonal influences on dimethylbenz(a)anthracene-induced tumor growth were investigated in detail by endocrine ablation and replacement of hormones. The majority of tumors regressed following ablation and most of them were reactivated by subsequent administrations of estrogen (0.1 to 5 mug) or prolactin (2 mg). Increasing numbers of tumors, however, were not stimulated by prolactin when administration was delayed, and a basal level of estradiol (0.01 mug) in addition to prolactin was required for reactivation of tumors. Nafoxidine hydrochloride, a competitor of estrogen at the receptor sites, arrested growth of a large portion of dimethylbenz(a)anthracene-induced tumors in intact animals but failed to retard growth of prolactin-stimulated tumors. On withdrawal of prolactin-nafoxidine, rapid regression of tumor occurred and readministration of prolactin failed to activate most of the tumors for as long as 28 days. Our results give good supporting evidence that estrogen plays a primary role in tumor growth. The interactions of prolactin and estrogen at tumor sites are necessary for regulatory events related to tumor growth.