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.     

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.     

Post-initiation treatment of rats with indole-3-carbinol or beta-naphthoflavone does not suppress 7, 12-dimethylbenz[a]anthracene-induced mammary gland carcinogenesis

Indole-3-carbinol (I3C) and β-naphthoflavone (β-NF), blocking agents of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mammary gland carcinogenesis, were examined as potential post-initiation suppressing agents. Treatment of female Sprague–Dawley rats with I3C (250 mg/kg body weight (b.w.)), β-NF (20 mg/kg b.w.) or the vehicle ethanol:corn oil (2:3) (2.5 ml/kg b.w.), three times weekly by gavage, started 3 weeks after the initiation with one oral dose of DMBA (20 mg/rat at 7 weeks of age) and continued for up to 12 weeks. I3C- or β-NF- or vehicle-treated groups did not differ significantly in the overall outcome of mammary tumorigenesis including cumulative mammary tumor incidences and multiplicities, latent periods and number and weight of mammary tumors per tumor-bearing rat for malignant, benign and/or malignant + benign tumors. A tendency of the I3C-treated rats to develop fewer mammary adenocarcinomas with a greater average weight per tumor per rat (2.32±1.50 g) than in the β-NF- (1.52±1.58 g) or vehicle- (1.55±1.53 g) treated groups suggests an effect, yet to be confirmed, of I3C on tumor development and growth. A 12-week treatment with I3C or β-NF significantly increased the P450-dependent activities of ethoxy-, methoxy-, benzyloxy- and pentoxy-(with I3C only) resorufin O-dealkylase in hepatic microsomes indicating induction of several P450s. The alterations in the P450 complement may affect endogenous estrogen metabolism and mammary gland and tumor characteristics at the molecular level, e.g. estrogen receptor status and/or proliferative activity, which require further studies.     

Inhibitory action of Piper betle on the initiation of 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis in rats

When an aqueous extract of the leaves of Piper betle, a medicinal plant, was given orally at different dose levels during the initiation phase of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis in rats, higher doses of the extract inhibited the emergence of tumors. However, when the extract was fed to the rats bearing DMBA-induced mammary tumors for 8 weeks, no appreciable degree of inhibition of tumor growth was noticed. Betel leaf extract at the dose levels used in the present study did not affect the body weight gain among rats.     

Effect of 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis on the opioid peptide levels in the rat central nervous system

Mammary tumors were induced in female Sprague-Dawley rats by giving a single oral dose of 20 mg 7,12-dimethylbenz[a]anthracene (DMBA). Animals were killed after full development of tumors 4 months after the ingestion of DMBA. Opioid peptides in various tissues were estimated by radioimmunoassay (RIA). Tumor-bearing rats (n = 5) had higher (P less than 0.05) contents of beta-endorphin in pituitary (+60%), striatum (+52%) and midbrain (+85%) compared to animals with no tumors. However, tumor-bearing rats showed a decrease of 35% in striatal met-enkephalin content. Dynorphin level decreased (P less than 0.05) in pituitary (-49%) and hypothalamus (-29%) of tumor-bearing rats. Thus for the first time, we report the alteration in the level of these neuropeptides during the process of chemical carcinogenesis.     

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.     

Inhibition of 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis by S-allylcysteine

Consumption of garlic has been reported to be associated with decreased risk of cancer. We used the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinoma model to assess the oral chemopreventive potential of S-allylcysteine (SAC), a water-soluble constituent of garlic. Hamsters were divided into four groups of six animals each. The right buccal pouches of the animals in group I were painted with a 0.5% solution of DMBA in liquid paraffin three times a week. The animals in group II were painted with DMBA as in group I and in addition received 200 mg/kg body weight SAC intragastrically three times a week on days alternate to DMBA application. Group III animals received SAC as in group II. Animals in group IV received neither DMBA nor SAC and served as control. The hamsters were killed after an experimental period of 14 weeks. Biochemical measurements were carried out on tumour and normal pouch tissues. Measurement of lipid peroxidation, reduced glutathione (GSH), glutathione peroxidase (GPx) and glutathione S-transferase (GST) was used to monitor the chemopreventive potential of SAC. All hamsters painted with DMBA alone for 14 weeks developed well-differentiated squamous cell carcinomas. Diminished lipid peroxidation in the oral tumour tissue was accompanied by a significant increase in the levels of GSH, GPx and GST. Administration of SAC significantly suppressed DMBA-induced oral carcinogenesis as revealed by the absence of neoplasms. The results of the present study suggest that garlic may exert its chemopreventive effects by modulating lipid peroxidation and enhancing the levels of GSH, GPx and GST.     

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.     

Telomerase activity during 7,12-dimethylbenz [a] anthracene-induced hamster buccal pouch carcinogenesis

Telomerase is a ribonucleoprotein complex intimately involved in cell immortalization and carcinogenesis. Increasing evidence shows that telomerase activity has been detected in human tumor cells but not in normal somatic tissues.[1] Thus, anhypothesis has been proposed that the activation of telomerase activity is essential for cells to overcome senescence and also may result in the immortality and malignant progression of a cancer.[2] Recently, comparatively low telomerase activity has been …     

Suppression of 7,12-dimethylbenz[alpha]anthracene-induced carcinogenesis and hypercholesterolaemia in rats by tocotrienol-rich fraction isolated from rice bran oil.

The anti-tumour and anti-cholesterol impacts of tocotrienol-rich fraction (TRF) were investigated in rats treated with the chemical carcinogen 7,12-dimethylbenz [alpha]anthracene (DMBA), which is known to induce mammary carcinogenesis and hypercholesterolaemia. DMBA administration to rats was associated with the appearance of multiple tumours on mammary glands after 6 months. Alkaline phosphatase (ALP) and glutathione-S-transferase (GST) are used as marker enzymes to monitor the severity of carcinogenesis. Although no tumours were visible on livers, hepatic ALP and GST activities of DMBA-treated rats were profoundly elevated in comparison to enzyme activities of normal control rats. Feeding of TRF (10 mg/kg body weight/day) for 6 months, isolated from rice bran oil (RBO), to DMBA-administered rats, reduced the severity and extent of neoplastic transformation in the mammary glands. Similarly, plasma and mammary ALP activities increased during carcinogenesis (95% and 43%, respectively), were significantly decreased in TRF-treated rats, whereas TRF mediated a further increase of 51% in hepatic ALP activity. TRF treatment to rats maintained low levels of GST activities in liver ( approximately 32%) and mammary glands ( approximately 21%), which is consistent with anti-carcinogenic properties of TRF. Administration of DMBA also caused a significant increase of 30% in plasma total cholesterol and 111% in LDL-cholesterol levels compared with normal control levels. Feeding of TRF to rats caused a significant decline of 30% in total cholesterol and 67% in LDL-cholesterol levels compared with the DMBA-administered rats. The experimental hypercholesterolaemia caused a significant increase in enzymatic activity (23%) and protein mass (28%) of hepatic 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase. Consistent with TRF-mediated reduction in plasma lipid levels, enzymatic activity and protein mass of HMG-CoA reductase was significantly reduced. These results indicate that TRF has potent anti-cancer and anti-cholesterol effects in rats.     

Fluorescence spectroscopic identffication of 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis

An attempt was made to study whether light-induced fluorescencespectroscopy could be exploited to discriminate premalignantand malignant tissues of hamster buccal pouch carcinogenesisfrom normal tissues during a 16 week regimen of tri-weekly topicalapplication of 7,12- dimethylbenz[a]anthracene (DMBA) in liquidparaffin. Histologically, the DMBA-treated buccal mucosa showedhyperplastic changes at 4–6 weeks, papillomas at 8–10weeks, early invasive carcinomas at 11–13 weeks and finallywell-differentiated squamous cell carcinomas at 14–16weeks of treatment. Acetone extracts of these different stagedtissues with age matched control tissues were excited at 405and 420 nm and the emissions were scanned from 430 and 440 to700 nm respectively. The spectral profiles of control and transformedtissues were found to be different, each displaying their owncharacteristic prominent maxima and other spectral marks. Thespectra of transformed tissues showed characteristic peaks around620–630 nm which did not appear in control tissues andthe fluorescent intensifies at 630 nm [FI(630)nm] were significantlyincreased from early stages onwards when compared to controls.The spectra of DMBA carcinomas developed at the 18th week afterwithdrawal of DMBA application at the 10th week and carcinomaextract spiked with DMBA conlirmed the peak around 620–630could be attributed only to porphyrin compounds accumulatedin transformed tissues. Furthermore, the ratios of FI(520)nm/FI(630)nm of transformed tissues were also significantly decreasedwhen compared to control tissues. This diagnostic test had avery close resemblance with respect to histological studies.These results suggest that this technique using conventionallight-induced fluorescence spectroscopy may be useful for earlydiagnosis of premalignant and malignant lesions of oral cavity.     

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.     

Garlic induces apoptosis during 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis

The apoptosis-inducing capacity of aqueous garlic extract during 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch (HBP) carcinogenesis was investigated in male Syrian hamsters using DNA fragmentation and the apoptosis-associated proteins, tissue transglutaminase (tTG) and Bcl-2. Hamsters were divided into four groups of six animals each. Animals in group 1 were painted with a 0.5% solution of DMBA in liquid paraffin on the right buccal pouches three times a week for 14 weeks. Group 2 animals painted with DMBA as in group 1, in addition received 250 mg/kg body weight aqueous garlic extract orally on days alternate to DMBA application. Group 3 animals received garlic extract as in group 2. Group 4 animals received neither DMBA nor garlic extract and served as the control. The experiment was terminated at the end of 14 weeks. Administration of aqueous garlic extract (250 mg/kg body weight) to animals painted with DMBA inhibited DMBA-induced oral carcinogenesis as revealed by the absence of neoplasms, induction of tTG and inhibition of Bcl-2 expression. The results of the present study suggest that garlic may exert its chemopreventive effect by inducing apoptosis.     

Modulation of 7,12-dimethylbenz[a]anthracene-induced transmammary carcinogenesis by disulfiram and butylated hydroxyanisole in mice

The individual as well as combined chemopreventive actions of disulfiram (DSF) and butylated hydroxyanisole (BHA) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced transmammary carcinogenesis in mice were examined. When nursing mothers receiving normal diet were treated with DMBA (1 mg/mouse) on days 6, 8 and 10 postpartum, the tumor incidence in their 50-week-old F1 progeny was 44.1%. When nursing mothers receiving 0.75% BHA diet, 0.5% DSF diet and 0.75% BHA + 0.5% DSF diet were similarly treated with DMBA, the tumor incidences in their 50-week-old F1 progeny were 14.7% (P less than 0.05), 12.5% (P less than 0.05) and 5.8% (P less than 0.01), respectively. It is concluded that diets containing BHA (0.75%) and DSF (0.5%), singly or in combination, can inhibit transmammary carcinogenesis in Swiss albino mice.     

Purple corn color suppresses Ras protein level and inhibits 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis in the rat

Anthocyanins belong to the class of phenolic compounds collectively named flavonoids. Many anthocyanins are reported to have inhibitory effects on carcinogenesis. Purple corn color (PCC), an anthocyanin containing extract of purple corn seeds, is used as a food colorant. The major anthocyanin in PCC is cyanidin 3- O -β- d -glucoside (C3-G). The present study was conducted to assess the influence of dietary PCC on 7,12-dimethylbenz[ a ]anthracene (DMBA)-induced mammary carcinogenesis in rats. PCC significantly inhibited DMBA-induced mammary carcinogenesis in human c-Ha- ras proto-oncogene transgenic (Hras128) rats and in their non-transgenic counterparts. PCC and C3-G also inhibited cell viability and induced apoptosis in mammary tumor cells derived from Hras128 rat mammary carcinomas. At the molecular level, PCC and C3-G treatment resulted in a preferential activation of caspase-3 and reduction of Ras protein levels in tumor cells. It is proposed that C3-G could act as a chemopreventive and possibly chemotherapeutic agent for cancers with mutations in ras . Secondly, the in vitro–in vivo system used in this study can be utilized for screening for cancer preventive compounds that act via Ras down-regulation. ( Cancer Sci 2008; 99: 1841–1846)     

Gene expression profiling in the mammary gland of rats treated with 7,12-dimethylbenz[a]anthracene

Identification of molecular markers of early-stage breast cancer development is important for the diagnosis and prevention of the disease. In the present study, we used microarray analysis to examine the differential expression of genes in the rat mammary gland soon after treatment with a known chemical carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA), and prior to tumor development. Six weeks after DMBA, differential expression of multiple genes involved in cell growth, differentiation and microtubule dynamics were observed. Gene expression changes were further validated by a combination of techniques, including real-time PCR, RT-PCR, Western blotting and immunohistochemistry. An inhibition of differentiation in this early stage was suggested by the lower expression of beta-casein and transferrin and higher expression of hsp27 in glands from DMBA-treated rats. Possible cell cycle deregulation was indicated by an increased expression of cyclin D1 and hsp86, a heat shock protein associated with cyclin D1. Prior to tumor development, DMBA increased cellular proliferation as detected by Ki-67 and stathmin immunostaining in histologically normal mammary gland. Genes regulating microtubule function, including stathmin, Ran, alpha-tubulin and hsp27, were all overexpressed in the mammary gland of DMBA-treated rats, raising the possibility that disruption of microtubule dynamics and abnormal mitosis may be critical events preceding breast cancer development. Several of the altered proteins, including hsp27, hsp86 and stathmin, may ultimately serve as markers of early breast cancer development.     

Post-initiation treatment of rats with indole-3-carbinol or β-naphthoflavone does not suppress 7,12-dimethylbenz[a]anthracene-induced mammary gland carcinogenesis

Indole-3-carbinol (I3C) and β-naphthoflavone (β-NF), blocking agents of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mammary gland carcinogenesis, were examined as potential post-initiation suppressing agents. Treatment of female Sprague–Dawley rats with I3C (250 mg/kg body weight (b.w.)), β-NF (20 mg/kg b.w.) or the vehicle ethanol:corn oil (2:3) (2.5 ml/kg b.w.), three times weekly by gavage, started 3 weeks after the initiation with one oral dose of DMBA (20 mg/rat at 7 weeks of age) and continued for up to 12 weeks. I3C- or β-NF- or vehicle-treated groups did not differ significantly in the overall outcome of mammary tumorigenesis including cumulative mammary tumor incidences and multiplicities, latent periods and number and weight of mammary tumors per tumor-bearing rat for malignant, benign and/or malignant + benign tumors. A tendency of the I3C-treated rats to develop fewer mammary adenocarcinomas with a greater average weight per tumor per rat (2.32±1.50 g) than in the β-NF- (1.52±1.58 g) or vehicle- (1.55±1.53 g) treated groups suggests an effect, yet to be confirmed, of I3C on tumor development and growth. A 12-week treatment with I3C or β-NF significantly increased the P450-dependent activities of ethoxy-, methoxy-, benzyloxy- and pentoxy-(with I3C only) resorufin O-dealkylase in hepatic microsomes indicating induction of several P450s. The alterations in the P450 complement may affect endogenous estrogen metabolism and mammary gland and tumor characteristics at the molecular level, e.g. estrogen receptor status and/or proliferative activity, which require further studies.     

Effects of selenium on 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis and DNA adduct formation

The purpose of the present investigation was to determine the effects of dietary selenium deficiency or excess on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary neoplasia in rats and to delineate whether selenium-mediated modification of mammary carcinogenesis was associated with changes in carcinogen:DNA adduct formation and activities of liver microsomal enzymes that are involved in xenobiotic metabolism. Female Sprague-Dawley rats were divided into three groups from weaning and were maintained on one of three synthetic diets designated as follows: selenium deficient (less than 0.02 ppm); selenium adequate (0.2 ppm); or selenium excess (2.5 ppm). For the DMBA binding and DNA adduct studies, rats were given a dose of [3H]DMBA p.o. after 1 month on their respective diets. Results from the liver and the mammary gland indicated that neither selenium deficiency nor excess had any significant effect on the binding levels, which were calculated on the basis of total radioactivity isolated with the purified DNA. Furthermore, it was found that dietary selenium intake did not seem to affect quantitatively or qualitatively the formation of DMBA:DNA adducts in the liver. Similarly, in a parallel group of rats that did not receive DMBA, the activities of aniline hydroxylase, aminopyrine N-demethylase, and cytochrome c reductase were not significantly altered by dietary selenium levels. Concurrent with the above experiments, the effect of dietary selenium intake on carcinogenesis was also monitored. Results of this experiment indicated that selenium deficiency enhanced mammary carcinogenesis only when this nutritional condition was maintained in the postinitiation phase. Likewise, an excess of selenium intake inhibited neoplastic development only when this regimen was continued after DMBA administration. In either case, deficient or excess selenium at the time of carcinogenic insult failed to produce a significant effect on subsequent tumor yield, if selenium intake was returned to normal during the proliferative phase of tumor growth. Based on the results of these studies, it is suggested that selenium-mediated modification of mammary tumorigenesis is not exerted via alterations in carcinogenic initiation (i.e., metabolism or DNA adduct formation).