Effect of the beta-diketones diferuloylmethane (curcumin) and dibenzoylmethane on rat mammary DNA adducts and tumors induced by 7,12- dimethylbenz[a]anthracene

Curcumin is a beta-diketone constituent of the spice turmeric that possesses anticarcinogenic properties in several animal models. The present studies were conducted in order to identify beta-diketones structurally-related to curcumin that would be effective dietary blocking agents toward the initiation stage of 7,12- dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinogenesis. Of the beta-diketone compounds initially screened for their capacity to induce quinone-reductase (QR) activity in wild-type Hepa1c1c7 cells and a mutant subclone, curcumin (diferuloylmethane) and dibenzoylmethane were most effective. However, when added to semipurified diets fed to female rats, dibenzoylmethane (1%), but not curcumin (1%), was effective in inhibiting in vivo mammary DMBA-DNA adduct formation. This inhibitory effect on mammary adduct formation was associated with a significant increase in liver activities of glutathione S-transferase, QR and 7-ethoxyresorufin-O-deethylase activities. Female rats provided diets supplemented with dibenzoylmethane at 0.1, 0.5 and 1.0% for 14 days prior to dosing with DMBA exhibited a significant decrease in mammary tumor development, compared with controls. However, tumor development for animals fed diets containing 1.0% curcumin was not different from that of controls. Therefore, dibenzoylmethane, and possibly other structurally-related beta-diketones, warrant examination as breast cancer chemopreventative blocking agents.      

Serum estrogens and estrogen responsiveness in 7,12-dimethylbenz[a]anthracene-induced mammary tumors as influenced by dietary fat

The effect of dietary fat on mammary tumor incidence, estrogen-binding capacity as related to the hormone dependency of the tumors, and circulating estrogen levels in Sprague-Dawley rats given an oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) was investigated. Rats were fed diets consisting of 0.5, 5, or 20% corn oil starting at weaning and were administered 5 mg DMBA at 50 days of age. Tumor incidences were 13, 46, and 75% for the groups given 0.5, 5, and 20% fat, respectively, when the experiment was terminated 20-22 weeks later. Serum estradiol, measured at proestrus at 50 days of age and at the end of the experiment, was slightly depressed at both time points in rats fed the 0.5% fat diet but was similar in the other 2 groups. Serum estrone levels were not significantly different at either time point. Estrogen receptor levels in the tumor were the same in the groups given 5 and 20% fat but were lower in the group given 0.5% fat. No difference was detected in the progesterone receptor concentrations. Furthermore, most (approximately 70%) of the tumors in all 3 dietary groups regressed in response to ovariectomy, which suggested that dietary fat has very little influence on the estrogen dependence of the tumor. This observation suggested that fat intake does not result in any intrinsic difference in the biochemical action of estrogen.     

The effect of dietary seaweeds on 7,12-dimethyl-benz[a]anthracene-induced mammary tumorigenesis in rats

Six groups of female rats were fed diets containing 2% of one of six powdered seaweeds for 152 days and a basic diet for 59 or 60 successive days, and controls were fed the basic diet for the whole experimental period. The 7,12-dimethylbenz[a]anthracene was given to all rats intragastrically (20 mg/kg X 1), 27 days after the start of feeding. Diets with 3 weeds, Porphyra tenera (PT), Laminaria religiosa (LR) and L. japonica var. ochotensis (LO), showed an inhibitory effect on mammary tumorigenesis. Tumor incidences were 35% (7/20), 35% (7/20) and 50% (9/18), respectively, whereas that in the control group was 69% (20/29). There was a significant delay in the time to first palpable tumor in LR-fed and PT-fed rats (P less than 0.01). As for the tumor weight per rat in each group, it was significantly lower in the LR-fed group with a weight of 1.6 g, as compared with that of 16.3 g in the control group (P less than 0.02).     

Serum cholesterol and 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis

Diet-induced changes in serum cholesterol levels and their relationship to mammary carcinogenesis initiated by 7,12-dimethylbenz[a]anthracene (DMBA) were studied in female Sprague-Dawley rats. DMBA was given to rats subjected to 3 dietary treatments: (1) a semipurified, cholesterol-free diet (SP); (2) the same diet with 1.5% cholesterol and 0.5% bile salts added (CB); (3) diet CB, until administration of DMBA and then switched to diet SP. Tumor yield per rat was increased in rats fed diet CB, but incidence and tumor size were similar among all 3 groups. Rats maintained on diet SP alone had a higher percentage of histologically benign tumors. Hypercholesterolemia of dietary origin appears to enhance slightly chemical carcinogenesis in this model.     

Doxorubicin-induced ultrastructural and growth changes in primary cultures of 7,12-dimethylbenz[a]anthracene-induced mammary tumors

For the determination of their response to doxorubicin (Dx) (adriamycin), monodispersed mammary tumor cells (from female outbred Sprague-Dawley rats) were maintained as monolayer primary culture. Dose-response curves and [3H]thymidine labeling indices showed the antimitotic and cytocidal effects of the drug varied in a dose-dependent fashion. Dose-response curves revealed that the median lethal dose concentration was 10(-4) M. A 24-hour treatment at concentrations of 10(-4) to 10(-5) M completely arrested DNA synthesis of the tumor cells. Surviving cells exhibited chromatin abnormalities, accumulation of cytoplasmic myelin bodies, vacuolization of endoplasmic reticulum, and increased density of mitochondrial matrix. This study showed 1) 7,12-dimethylbenz[a]anthracene-induced mammary tumor cells were highly sensitive to Dx; 2) Dx induced specific ultrastructural effects on the nuclei, mitochondria, and membranes of the cells; and 3) the in vitro response of the primary culture may be useful for prediction of the response of the source tumor to chemotherapy.     

Effect of naturally occurring coumarins on the formation of epidermal DNA adducts and skin tumors induced by benzo[a]pyrene and 7,12- dimethylbenz[a]anthracene in SENCAR mice

Several naturally occurring coumarins previously found to be potent inhibitors of mouse hepatic ethoxyresorufin-O-deethylase (EROD) and/or pentoxyresorufin-O-dealkylase (PROD) were examined for their effects on formation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) DNA adducts in mouse epidermis, as well as, their effects on skin tumor initiation by these polycyclic aromatic hydrocarbons (PAH). Bergamottin, a potent inhibitor of hepatic EROD, given topically 5 min prior to an initiating dose of B[a]P, significantly decreased total covalent binding of B[a]P to DNA in a dose-dependent manner 24 h after treatment. A dose of 400 nmol bergamottin reduced covalent binding of B[a]P by 72%. Coriandrin, at a dose of 400 nmol also significantly reduced total covalent binding of B[a]P by 59%. In addition, formation of the major (+)anti-B[a]P-diol epoxide-N2-dGuo adduct was selectively reduced by both of these coumarins. In contrast, bergamottin and coriandrin did not significantly decrease covalent binding of DMBA to epidermal DNA at doses of either 400 nmol or 800 nmol. Imperatorin and isopimpinellin, which are more potent inhibitors of hepatic PROD activity, significantly reduced overall binding of DMBA to epidermal DNA by 67% and 52%, respectively, when applied at doses of 400 nmol. These two coumarins also inhibited B[a]P-DNA adduct formation at similar doses but to a lesser extent. Imperatorin at a dose of 400 nmol dramatically decreased formation of covalent DNA adducts derived from both the anti and syn diol epoxides of DMBA. Bergamottin was a potent inhibitor of tumor initiation by B[a]P while coriandrin was less effective in this regard. Imperatorin was an effective inhibitor of skin tumor initiation by DMBA and also inhibited complete carcinogenesis by this PAH. At dose levels higher than those effective against DMBA, imperatorin also inhibited tumor initiation by B[a]P. The results demonstrate that several naturally occurring coumarins possess the ability to block DNA adduct formation and tumor initiation by PAHs such as B[a]P and DMBA. The mechanism for reduced DNA adduct formation and tumor initiation appears to involve inhibition of the P450s involved in the metabolic activation of these hydrocarbons. Finally, the differential effects of certain coumarins on B[a]P vs DMBA DNA adduct formation and tumor initiation may be useful for dissecting the role of specific cytochromes P450 in their metabolic activation.      

Selenium-mediated inhibition of 7,12-dimethylbenz[a]anthracene-induced mouse mammary tumorigenesis

The effect of supplemental selenium on 7,12-dimethyl-benz[a]anthracene(DMBA)-induced mammary tumori-genesis was investigated in severalmouse strains. Selenium, administered as SeO₂ in the drinkingwater, inhibited mammary tumor formation in DMBA-treated (C57BLx DBA/2f)F₁, C3H/StWi and BALB/c female mice. In addition, seleniuminhibited the occurrence of DMBA-induced ductal hyperplasiasin (C57BL x DBA/2f)F₁ and BALB/c mice and mammary tumor virus-inducedalveolar hyperplasias in BALB/cfC3H mice. Selenium did not alterthe growth of established mammary tumors. These results demonstratethat supplemental selenium inhibits both chemical- and viral-inducedmouse mammary tumorigenesis, and secondly, that the developmentof preneoplastic lesions, an early stage in mammary tumorigenesis,is very sensitive to selenium-mediated inhibition.     

Inhibition of 7,12-dimethylbenz[a]anthracene-induced mammary tumors and DNA adducts by garlic powder.

The present studies determined the influence of dietary supplements of garlic powder (0, 1, 2 or 4%) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors and on the in vivo occurrence of mammary DMBA-DNA adducts in rats. Diets were offered 2 weeks before and 2 weeks following DMBA treatment (25 mg/kg body wt). An additional group was fed the 2% garlic powder diet throughout the 20 week study. Although food intake and weight gain were not influenced, dietary garlic powder supplementation did significantly delay the onset of first tumors (P < 0.01) and did reduce the final mammary tumor incidence (P < 0.01). Consumption of garlic powder also significantly depressed the in vivo binding of DMBA to mammary cell DNA. Binding of both anti- and syn-dihydrodiol epoxides to DNA was depressed in rats fed supplemental garlic powder. The activity of glutathione S-transferase (GST) in mammary and liver tissue from rats fed 2% dietary garlic powder was higher than observed in tissues from rats fed the basal diet. No further increase in GST activity occurred when the dietary garlic content was increased from 2 to 4%. Final mammary tumor incidence was found to correlate positively with total DMBA-DNA binding and the quantities of individual DMBA-DNA adducts. The present studies demonstrate that garlic powder is effective in inhibiting DMBA-induced mammary tumors, possibly by reducing DMBA-DNA binding.     

Effect of n-3 and n-6 fatty acids on 7,12 dimethylbenz (a) anthracene-induced mammary tumorigenesis

Numerous studies have consistently shown that vegetable oils containing linoleic acid enhance mammary tumorigenesis more effectively than fish oils containing eicosapentaenoic and docohexaenoic acids. The purpose of this investigation was to study these and additional n-3 and n-6 PUFA, e.g., a-linolenic (a-LN) (18:3 n-3) and gamma-linolenic (GLA) (18:3 n-6) acid. Different oils were used as dietary sources of fatty acids: corn (CO) (61% LA); blackcurrant (BCO) (44% LA, 18% GLA and 16% a-LN); fish oil (FO) (mixed with corn oil, 12% LA and 24% EPA + DPA + DHA). Thirty-five-day-old female Sprague-Dawley rats were divided into 5 dietary treatment groups and were allowed to feed ab libitum on one of the test diets: I. BCO (23.5%); II. CO (23.5%); III. BCO (15.5%) + FO (8%); IV. FO (20.5%) + CO (3%); and V. BCO (20.5%) + FO (3%). From 48 to 52 days of age, rats in all five groups were fed rat chow. At 50 days of age, all rats were given 5 mg DMBA by oral intubation, and 2 days later the test diets were resumed until termination of the experiment. Analysis of tumor incidence, and multiplicity data for 5 diet groups indicated that rats fed 23.5% CO (II) exhibited enhanced mammary tumor yields when compared to animals on the remaining 4 diets in the order II greater than I, III, V greater than IV. Since the level of fat (23.5% w/w) was similar in all 5 diets, and body weight gain was in the order IV greater than II greater than I, the results of this study indicate that differences in tumor yields were related to fatty acid composition of diets. In support of this conclusion, fatty acid profiles of RBC and tumor phosphoglycerides reflected dietary fatty acid composition. In groups I and II, even though tumor levels of LA were similar, the levels of GLA, DHLA (20:3 n-6) and a-LN were higher in I compared to II, suggesting that these differences may be associated with lower yields of DMBA-induced mammary tumors in group I. Incorporation of marine type n-3 PUFA (EPA, DPA and DHA) in tumor PL was greater in Group IV compared to plant type n-3 PUFA (a-LN) in Groups I, III, and V. Since tumor yields were the lowest in Group IV, these results suggest that incorporation of marine type n-3 PUFA into cell membranes does not favor development of DMBA-induced mammary tumors.     

Inhibition of 7,12-dimethylbenz(a)anthracene-induced mammary tumors and DNA adducts by dietary selenite

The present studies were designed to examine the influence of dietary selenite supplementation on the initiation phase of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis and to correlate selenite-induced changes in the binding of DMBA metabolites to rat mammary cell DNA with the ultimate tumor incidence. Diets formulated to contain selenium, as sodium selenite at 0.1, 0.5, 1, 2, or 4 micrograms/g were fed for 2 weeks prior to and 2 weeks following treatment with DMBA (5 mg/kg body weight). Food intake and weight gain did not differ among treatments. Tumor incidence correlated inversely to the quantity of selenium consumed (r = -0.99). Final tumor incidences were 52, 32, 24, 14, and 10% for rats fed 0.1, 0.5, 1, and 4 micrograms selenium/g, respectively. In a separate group of rats fed a diet containing 4 micrograms selenium/g during both the initiation and promotion stages the final tumor incidence was 4.8%. Selenite supplementation for 2 weeks markedly depressed the occurrence of individual and total DMBA-DNA adducts. The final mammary tumor incidence correlated positively with total DMBA-DNA adducts (r = 0.99). These studies clearly demonstrate that selenite can inhibit the initiation stage of mammary carcinogenesis. This reduction in tumor incidence is likely due to a reduction in carcinogen metabolism and ultimately adduct formation.     

Restraint-induced inhibition of 7,12-dimethylbenz[a]anthracene-induced mammary tumors: relation to stages of tumor development.

Experiments on the use of chronically applied restraint to reduce the number of mammary tumors developing in response to treatment of rats with 7,12-dimethylbenz[a]anthracene indicated that this inhibitory effect was largely due to restraint applied after the induction period; preinduction restraint and induction period restraint had no significant effect on tumor development. After termination of the restraint treatment, the rate at which new tumors appeared first increased and then decreased. Restraint did not affect the proportion of tumors regressing after ovariectomy.     

Regulation of cyclic adenosine 3',5'-monophosphate in relation to growth of dimethylbenz[a]anthracene-induced mammary tumors in rats

In this study, adenylate cyclase, phosphodiesterase and cyclic adenosine 3',5'-monophosphate (cAMP) levels were measured in 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors with different growth characteristics in both intact and ovariectomized rats. Tumors were classified as growing, stable, or regressing over a 10-14-day period before excision. Regressing or static tumors had a higher cAMP concentration relative to tumors that were growing actively. This was due to high adenylate cyclase activity and low phosphodiesterase (both high and low Km) activities. Although estrogen deprivation resulted in a much greater rate of tumor regression than would occur spontaneously under normal conditions, the levels of adenylate cyclase, phosphodiesterases and cAMP were the same in tumors obtained from either intact or ovariectomized rats, when comparisons were made within the same category of tumor. These observations suggest that cAMP metabolism is independent of estrogen, since it is related to enhancement or retardation of growth rather than to the presence of absence of estrogen.     

Inhibition of N-methyl-N-nitrosourea- and 7,12-dimethylbenz[a] anthracene-induced rat mammary tumorigenesis by dietary cholesterol is independent of Ha-Ras mutations

Dietary cholesterol has previously been shown to inhibit rat mammary tumorigenesis but the mechanisms remain unclear. Uptake of serum low density lipoprotein cholesterol by tissues leads to down-regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis that catalyzes the formation of mevalonate. In addition to being a precursor of cholesterol, mevalonate is necessary for DNA synthesis and cell proliferation. Isoprenoids, also derived from mevalonate, are required for the post-translational modification of Ras proteins that are mutated in a number of carcinogen-induced rat mammary tumors. The purpose of this study, therefore, was to determine whether inhibition of tumorigenesis by cholesterol is dependent on the frequency of mutations in the Ha-ras gene. Female Sprague-Dawley rats (30/group) were given a single dose of either N-methyl-N-nitrosourea (MNU, 50 mg/kg i.p.) or 7, 12-dimethylbenz[a]anthracene (DMBA, 100 mg/kg intragastrally), carcinogens that produce tumors with either a high (MNU) or low (DMBA) frequency of Ha-ras mutations in codon 12 or 61, respectively. Rats were fed either a control AIN-93G diet or the control diet supplemented with 0.3% cholesterol for 14 weeks. Dietary cholesterol significantly decreased the final tumor incidence in rats given DMBA (83 versus 100%, P < 0.05) or MNU (53 versus 77%, P < 0.05). HMG-CoA reductase activity was higher in mammary tumors than in normal mammary glands, but the activity of this enzyme was reduced by cholesterol feeding only in mammary glands and not in tumors. Tumors induced by MNU had a high frequency of Ha-ras mutations in both the control (65%) and cholesterol-fed (68%) groups. Tumors induced by DMBA had a low frequency of Ha-ras mutations that also did not differ between the control (21%) and cholesterol-fed (18%) groups. These findings show that dietary cholesterol inhibits mammary tumorigenesis induced by either MNU or DMBA and that the inhibition is independent of the type or extent of mutations in the Ha-ras gene.     

Effects of high dietary fat on the total DNA and receptor contents in rats with 7,12-dimethylbenz[a]anthracene-induced mammary carcinoma

The influence of high dietary fat on the malignant intensity and the hormone receptors of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinoma in female Sprague-Dawley rats were analyzed by the tumor incidence and growth, the DNA histogram type, the DNA index, the S-phase fraction, and the estrogen receptor (ER) and progesterone receptor (PgR) assays. The rats were fed either a low-fat (0.5% corn oil) diet or a high-fat (20% corn oil) diet after the DMBA administration. Tumor incidences in the low-fat and the high-fat diet groups were 46 and 86%, respectively (p less than 0.01). Tumors in the high-fat diet group were also significantly larger than those in the low-fat group. Average tumor latent period was significantly shorter in the high-fat diet group, comparing with that in the low-fat diet group (p less than 0.01). Sixty-nine percent of the tumors in the high-fat diet group had aneuploid type, while only 8% of those in the low-fat diet group had aneuploid type. The DNA index and S-phase fraction also were significantly higher in the high-fat diet group (p less than 0.01). But the ER and PgR contents were not different between both groups. Therefore, these results suggest that a high dietary fat could increase the malignant intensity of the tumor but does not influence the hormonal responsiveness of these tumors.     

Beta-carotene inhibition of 7,12-dimethylbenz[a]anthracene-induced transformation of murine mammary cells in vitro

The chemopreventive action of ß-carotene during chemically-inducedtransformation of the epithelial cells in organ culture of thewhole mammary glands from BALB/c female mice was studied. Themammary epithelial cells in the whole mammary organ in a hormonesupplemented, serum-free medium were transformed after 24 hexposure to 7.8 µM 7,12-dimethylbenz[a]anthracene (DMBA)between 3rd and 4th day of a total 10 day culture period. Thetransformation process was associated with appearance of nodule-likealveolar lesions (NLAL) in glands in vitro. The epithelial cellstransformed by DMBA are potentially neoplastic, and NLAL servesas a morphological marker of preneoplasia in the glands in vitro.Treatment with ß-carotene (10^–6 M) during DMBAexposure (3rd – 4th day) caused 68% inhibition in thenumber of glands with incidence of NLAL. A 49% inhibition ofNLAL was evident when the glands were incubated with ß-carotene(days 4–10) after exposure to DMBA. Results indicate thatß-carotene inhibits DMBA-induced transformation ofthe mammary glands in vitro acting both at the initiation andthe promotional stages. This inhibitory effect is likely dueto the action of ß-carotene itself since no accumulationof retinol, the metabolic derivative of the vitamin A precursor,was detectable in the mammary glands during the 10 day cultureperiod.