Ellagic acid coordinately attenuates Wnt/ß-catenin and NF-κB signaling pathways to induce intrinsic apoptosis in an animal model of oral oncogenesis.

PURPOSE: Constitutive activation of the Wnt signaling pathway and its downstream effectors plays a key role in neoplastic transformation. The objective of this study was to investigate the effect of ellagic acid, a plant-derived polyphenol on Wnt/ß-catenin signaling and its downstream circuits- NF-κB and mitochondrial apoptosis in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model. METHODS: Hamsters were divided into six groups. The right buccal pouches of animals in groups 1-4 were painted with 0.5% DMBA three times a week for 14 weeks. Animals in groups 2-4 received in addition basal diet containing ellagic acid at a concentration of 0.1, 0.2, and 0.4% in the diet. Group 5 animals were given 0.4% ellagic acid alone. Group 6 animals served as control. The expression of the members of Wnt and NF-κB signaling and intrinsic apoptosis was evaluated by western blot analysis. RESULTS: Dietary supplementation of 0.4% ellagic acid suppressed the development of HBP carcinomas by preventing the constitutive activation of Wnt pathway through the downregulation of Fz, Dvl-2, GSK-3ß and nuclear translocation of ß-catenin. Abrogation of Wnt signaling by ellagic acid was also associated with inactivation of NF-κB and modulation of key components of the mitochondrial apoptotic network. CONCLUSIONS: Our findings suggest a functional crosstalk between Wnt and NF-κB signaling pathways in HBP carcinomas that is blocked by ellagic acid supplementation. Dietary ellagic acid that targets the Wnt/ß-catenin pathway as well as its downstream signaling mediators is a unique candidate for cancer chemoprevention.

Method validation and simultaneous determination of retinol, retinyl palmitate, ß-carotene, α-tocopherol and vitamin C in rat serum treated with 7,12 dimethylbenz[a]anthracene and Plantago major L. by high- performance liquid chromatography using diode-array detection.

A new and simple high-performance liquid chromatography method was developed and validated for the simultaneous determination of retinol, retinyl palmitate, ß-carotene, α-tocopherol and vitamin C in rat serum treated with Plantago Major L. and 7,12 dimethylbenz[a]anthracene. High-performance liquid chromatography analysis was performed utilizing an Inertsil ODS3 reversed phase column with methanol-tetrahydrofuran-water as mobile phase under gradient conditions, at 1.5 mL min(-1) flow rate and 25 °C. Diode-array detection was at 325, 450, 290 and 270 nm (retinol and retinyl palmitate), ß-carotene, α-tocopherol and vitamin C, respectively and runnig time 18 min. The high-performance liquid chromatography assay and extraction procedure proposed are simple, rapid, sensitive and accurate. The method was then applied for the determination of retinol, retinyl palmitate, ß-carotene, α-tocopherol and vitamin C in rat serum. Results of this study demonstrated that; at 60th day DMBA-treated group, there was a significant decrease in vitamin levels compared to the levels of control group. A significant increase was observed in vitamin levels of 7,12 dimethylbenz[α]anthracene+Plantago Major L.-treated group compared to the DMBA-treated group. Additionally, the results obtained in the study are found to be in agreement with data reported in the literature.

Synergistic growth inhibition of mouse skin tumors by pomegranate fruit extract and diallyl sulfide: evidence for inhibition of activated MAPKs/NF-κB and reduced cell proliferation.

Limited outcomes from earlier chemopreventive studies have necessitated that some modifications be made to get better efficacy. It is proposed that cancer prevention is more feasible than treatment, and this could be achieved effortlessly with use of multiple agents competent of targeting multiple targets. This study was initiated to examine the chemopreventive efficacy of pomegranate fruit extract (PFE) and diallyl sulfide (DAS), alone and in combination, using 2-stage mouse skin tumorigenesis model. PFE and DAS alone delayed onset and tumor incidence by ∼55% and ∼45%, respectively, while their combination at low doses synergistically decreased tumor incidence more potentially (∼84%, p<0.01). In addition, regression in tumor volume was seen with continuous combinatorial treatment (p<0.01). Mechanistic studies revealed that this inhibition was associated with decreased expression of phosphorylated ERK1/2, JNK1 and activated NF-κB/p65, IKKα, IκBα phosphorylation and degradation in skin tissue/tumor. Histological and cell death analysis also confirmed that combined PFE and DAS inhibit cellular proliferation and markedly induce apoptosis than the single agents. Altogether, our results suggest that PFE and DAS in combination impart better suppressive activity than either of these agents alone and provide support that development of novel combination therapies/chemoprevention using dietary agents will be more beneficial against cancer.

Raising gestational choline intake alters gene expression in DMBA-evoked mammary tumors and prolongs survival.

Choline is an essential nutrient that serves as a donor of metabolic methyl groups used during gestation to establish the epigenetic DNA methylation patterns that modulate tissue-specific gene expression. Because the mammary gland begins its development prenatally, we hypothesized that choline availability in utero may affect the gland's susceptibility to cancer. During gestational days 11-17, pregnant rats were fed a control, choline-supplemented, or choline-deficient diet (8, 36, and 0 mmol/kg of choline, respectively). On postnatal day 65, the female offspring received 25 mg/kg of a carcinogen 7,12-dimethylbenz[alpha]anthracene. Approximately 70% of the rats developed mammary adenocarcinomas; prenatal diet did not affect tumor latency, incidence, size, and multiplicity. Tumor growth rate was inversely related to choline content in the prenatal diet, resulting in 50% longer survival until euthanasia, determined by tumor size, of the prenatally choline-supplemented rats compared with the prenatally choline-deficient rats. This was accompanied by distinct expression patterns of approximately 70 genes in tumors derived from the three dietary groups. Tumors from the prenatally choline-supplemented rats overexpressed genes that confer favorable prognosis in human cancers (Klf6, Klf9, Nid2, Ntn4, Per1, and Txnip) and underexpressed those associated with aggressive disease (Bcar3, Cldn12, Csf1, Jag1, Lgals3, Lypd3, Nme1, Ptges2, Ptgs1, and Smarcb1). DNA methylation within the tumor suppressor gene, stratifin (Sfn, 14-3-3sigma), was proportional to the prenatal choline supply and correlated inversely with the expression of its mRNA and protein in tumors, suggesting that an epigenetic mechanism may underlie the altered molecular phenotype and tumor growth. Our results suggest a role for adequate maternal choline nutrition during pregnancy in prevention/alleviation of breast cancer in daughters.

Purple grape juice inhibits 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumorigenesis and in vivo DMBA-DNA adduct formation.

There has been considerable interest in identifying specific foods and phytochemicals that may have breast cancer preventive properties. Concord grapes are rich in polyphenolic chemicals and anthocyanin pigments that may have biological properties which could suppress cancer such as having antioxidant, antiproliferative, and proapoptotic actions. To determine the potential breast cancer protective action of purple grape juice, we examined the effect of grape juice consumption on the initiation stage of 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumorigenesis and on the in vivo formation of rat mammary DNA adducts in female Sprague-Dawley rats. Consumption of grape juice significantly inhibited mammary tumor mass at termination and the growth of tumors for the first 5 weeks of detectable tumor development. Consumption of grape juice phenolics by rats also significantly inhibited in vivo mammary DMBA-DNA adduct formation by 34 and 56% for animals fed phenolics at 346 and 692 mg/dL, respectively, compared to controls. Mammary 8-oxo-deoxyguanosine (8-oxo-dG) levels decreased by 25 and 37%, respectively, but the differences were not statistically significant. Liver DMBA-DNA adducts decreased by 10-30%, while 8-oxo-dG adducts remained unchanged, following grape juice intake. Liver glutathione S-transferase activity was significantly increased following grape juice consumption, but only at the highest level of intake. In addition, liver activities of catalase increased and xanthine oxidase decreased significantly, but only at the highest grape juice dose. Thus, these studies indicate that specific constituents or combinations of phytochemicals in purple grape juice can block the initiation stage of DMBA-induced rat mammary tumorigenesis. This tumor inhibitory effect was associated with a suppression of mammary DMBA-DNA adduct formation, which in part may be explained by increased liver activity of the phase II metabolizing enzyme, glutathione S-transferase. Mammary and liver 8-oxo-dG levels were not significantly altered by grape juice consumption. Thus, grape juice constituents appear to have benefit in decreasing susceptibility of the rat mammary gland to the tumor-initiating action of DMBA.

Comparative action of 1,4-phenylenebis(methylene)selenocyanate and its metabolites against 7,12-dimethylbenz[a]anthracene-DNA adduct formation in the rat and cell proliferation in rat mammary tumor cells.

1,4-phenylenebis(methylene)selenocyanate (p-XSC) inhibits 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis and DMBA-DNA binding in the rat mammary gland. Tetraselenocyclophane (TSC) was identified in rat feces as a metabolite of p-XSC. This led us to postulate the metabolic pathway: p-XSC-->glutathione conjugate (p-XSeSG)-->aromatic selenol (p-XSeH)-->TSC. Whether p-XSC or one of its metabolites is responsible for cancer prevention is the focus of this study. We utilized the DMBA-DNA binding assay with p-XSC as a positive control to evaluate the chemopreventive potential of p-XSC metabolites at dietary selenium levels of 10 ppm. Rats were fed AIN-76A diet supplemented with various selenium compounds for 1 week prior to the oral administration of a single dose of [3H]DMBA (5 mg per rat, specific activity 51.3 mCi/mmol). The rats were sacrificed 24 h later and DNA was isolated from the mammary fat pads. Relative levels of total binding were: [pmol/mg DNA, mean +/- S.D., n=6]; DMBA [7.2 +/- 1.6]; DMBA+p-XSC [3.5 +/- 2.7]; DMBA+p-XSeSG [2.2 +/- 1.1]; DMBA+TSC [5.6 +/- 2.9]. All selenium compounds, except TSC, significantly inhibited DMBA-DNA adduct formation; however, the difference between p-XSC and p-XSeSG was not statistically significant. The inhibition of total binding was attributed to a reduction in the formation of the three major adducts derived from bay-region diol epoxides of DMBA. On the basis of their chromatographic characteristics, these were identified as anti-diol-epoxide:deoxyguanosine, syn-diol-epoxide:deoxyadenosine, and anti-diol-epoxide:deoxyadenosine. Our results suggest that p-XSeSG, but not TSC, is the likely inhibitor of mammary cancer. Selenium levels measured by atomic absorption spectroscopy in the target organ (mammary fat pads) and in plasma following the dietary administration of selenium compounds were in the order of p-XSeSG congruent with p-XSC>TSC. These results appear to be consistent with their order of inhibitory effects on total DMBA-DNA binding. Further in vitro studies of the effect of selenium compounds on cell proliferation suggest that, depending on the dose and time point selected, p-XSC is comparable to or better than p-XSeSG; but both are more effective than TSC. Collectively, our in vivo and in vitro results indicate that p-XSC and its conjugate are better candidates than TSC for future studies on mammary cancer chemoprevention.

The red clover (Trifolium pratense) isoflavone biochanin A modulates the biotransformation pathways of 7,12-dimethylbenz[a]anthracene.

Several flavonoids have shown their anti-carcinogenic effects in various models. The soyabean isoflavone genistein was demonstrated earlier in our laboratory to be an effective inhibitor of dimethylbenz[a]anthracene (DMBA)-induced DNA damage in MCF-7 cells by curbing cytochrome P450 (CYP) 1 enzymes. The red clover (Trifolium pratense) isoflavone biochanin A is a methylated derivative of genistein, and its anti-mutagenic effect in bacterial cells has been shown previously. Because of its protection against chemical carcinogenesis in an animal model, biochanin A was selected for testing in our established MCF-7 cell system. From the results obtained in the semi-quantitative reverse transcription-polymerase chain reaction and xenobiotic response element (XRE)-luciferase reporter assays, biochanin A could reduce xenobiotic-induced CYP1A1 and -1B1 mRNA abundances through the interference of XRE-dependent transactivation. Enzyme kinetic studies also indicated that biochanin A inhibited both CYP1A1 and -1B1 enzymes with inhibition constant (Ki) values 4.00 and 0.59 microm respectively. Since the biotransformation of DMBA was dependent on CYP1 enzyme activities, biochanin A was able to decrease the DMBA-DNA lesions. The present study illustrated that the red clover isoflavone could protect against polycylic aromatic hydrocarbon-induced DNA damage.

Inhibition by ajoene of skin-tumor promotion in mice.

Ajoene, a major compound containing sulfur in oil-macerated garlic products, inhibited in a two-stage carcinogenesis test on mouse skin. Treatment with ajoene suppressed skin tumor formation, depending on the amount. In particular, the group treated with 250 microg of ajoene had only 4.9% the number of tumors per mouse compared with the control group at 18 weeks.

Cafestol and kahweol, two coffee specific diterpenes with anticarcinogenic activity.

Epidemiological studies have found an inverse association between coffee consumption and the risk of certain types of cancers such as colorectal cancers. Animal data support such a chemopreventive effect of coffee. Substantial research has been devoted to the identification of coffee components that may be responsible for these beneficial effects. In animal models and cell culture systems, the coffee diterpenes cafestol and kahweol (C+K) were shown to produce a broad range of biochemical effects resulting in a reduction of the genotoxicity of several carcinogens including 7,12-dimethylbenz[a]anthracene (DMBA), aflatoxin B(1) (AFB(1)), benzo[a]pyrene (B[a]P) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Different mechanisms appear to be involved in these chemoprotective effects: an induction of conjugating enzymes (e.g. glutathione S-transferases, glucuronosyl S-transferases), an increased expression of proteins involved in cellular antioxidant defense (e.g. gamma-glutamyl cysteine synthetase and heme oxygenase-1) and an inhibition of the expression and/or activity of cytochromes P450 involved in carcinogen activation (e.g. CYP2C11, CYP3A2). In animal models, the C+K-mediated induction of conjugating and antioxidant enzymes has been observed in hepatic, intestinal and kidney tissues. In the small intestine, these inductions were shown to be mediated by Nrf2-dependent transcriptional activation. In vitro investigations obtained in cell cultures of human origin indicate that the effects and mechanisms observed in animal test systems with C+K are likely to be of relevance for humans. In human liver epithelial cell lines transfected to express AFB(1)-activating P450s, C+K treatment resulted in a reduction of AFB(1)-DNA binding. This protection was correlated with an induction of GST-mu, an enzyme known to be involved in AFB(1) detoxification. In addition, C+K was found to inhibit P450 2B6, one of the human enzymes responsible for AFB(1) activation. Altogether, the data on the biological effects of C+K provide a plausible hypothesis to explain some of the anticarcinogenic effects of coffee observed in human epidemiological studies and in animal experiments.

Role of cytochrome P450 1a1 and 1b1 in the metabolic activation of 7,12-dimethylbenz[a]anthracene and the effects of naturally occurring furanocoumarins on skin tumor initiation.

The current study was designed to determine the mechanistic basis for differences in the effects of naturally occurring furanocoumarins on skin tumor initiation by 7,12-dimethylbenz[a]anthracene (DMBA). Female SENCAR mice were pretreated topically with bergamottin, imperatorin, or isopimpinellin (100-3200 nmol), 7,8-benzoflavone (7,8-BF, 5-40 nmol, a known inhibitor of DMBA skin carcinogenesis in mice), or acetone (vehicle control) 5 min prior to topical treatment with DMBA (10 nmol). Imperatorin, isopimpinellin, and 7,8-BF, but not bergamottin, significantly blocked total DMBA-DNA adduct formation. HPLC analysis of DNA adducts revealed that bergamottin preferentially inhibited formation of anti-DMBA diol-epoxide (DMBADE) derived DNA adducts, imperatorin, and isopimpinellin inhibited both anti- and syn- derived adducts, whereas 7,8-BF showed some selectivity for reduction of syn-DMBADE-DNA adducts. Mouse embryo fibroblast C3H/10T1/2 (10T1/2) cells, and mouse hepatoma-derived 1c1c7 (Hepa-1) cells, which preferentially express P450 1b1 and P450 1a1, respectively, were co-incubated with 2 microM bergamottin, imperatorin, isopimpinellin, and 7,8-BF, and with DMBA (2 microM). Hepa-1 cells (P450 1a1) formed mainly anti-DMBADE-DNA adducts. In contrast, 10T1/2 cells (P450 1b1) formed mainly syn-DMBADE-DNA adducts. Bergamottin inhibited DMBA metabolism to DMBA-3,4-diol and blocked DNA adduct formation in Hepa-1 cells, but had little effect in 10T1/2 cells. In contrast, 7,8-BF completely blocked DMBA metabolism and DNA adduct formation in 10T1/2 cells, but had little effect in Hepa-1 cells. Imperatorin and isopimpinellin inhibited DMBA bioactivation in both cell lines. These results indicate that bergamottin is a more selective inhibitor of P450 1a1 and overall a less effective inhibitor of the metabolic activation of DMBA in mouse epidermis. In contrast, imperatorin, isopimpinellin, and especially 7,8-BF, which block metabolic activation of DMBA in mouse epidermis, appear more selective for P450 1b1. On the basis of our studies using 10T1/2 cells and Hepa-1 cells, it appears that P450 1a1 is primarily responsible for converting DMBA-3,4-diol to anti-DMBADE, whereas P450 1b1 is primarily responsible for converting DMBA-3,4-diol to syn-DMBADE. These data demonstrate the role of P450 1a1 and 1b1 in the metabolic activation of DMBA in mouse epidermis and provide a mechanistic explanation for the differential effects of naturally occurring furanocoumarins (and 7,8-BF) on polycyclic aromatic hydrocarbon skin carcinogenesis.

Effect of dietary soy protein isolate, genistein, and 1,4-phenylenebis(methylene)selenocyanate on DNA binding of 7,12-dimethylbenz[a]anthracene in mammary glands of CD rats.

We examined whether a soy protein isolate or one of its major components (genistein) influences the initiation stage of carcinogenesis via DNA binding studies of 7,12-dimethylbenz[a]anthracene (DMBA) in liver and mammary tissue of female CD rats. A semipurified high-fat diet (23.5% corn oil) containing the soy protein isolate (10%), genistein (111 ppm), or 1,4-phenylenebis(methylene)selenocyanate (p-XSC) (5 ppm as selenium) as a positive control was fed to 6-week-old virgin female CD rats for 1 week before carcinogen treatment. Neither soy nor genistein affected the extent of DMBA-DNA binding in liver. In mammary tissue, 111 ppm genistein in the diet was more effective than the soy protein isolate, although the latter contains the same amount of genistein, mainly present as a glucoside conjugate. As shown before, p-XSC inhibited DMBA-DNA binding in mammary tissue. Total binding was inhibited because of reduced formation of three major adducts: anti-diol epoxide deoxyguanosine, syn-diol epoxide deoxyadenosine, and anti-diolepoxide deoxyadenosine. Thus, an additional experiment with 111 and 222 ppm of genistein was performed; 222 ppm genistein had a weaker effect than that observed for 111 ppm. Nevertheless, 111 ppm of genistein in the diet appears to inhibit the initiation phase of DMBA-induced rat mammary tumors and may partially account for the reported inhibitory effect of soy against DMBA-induced rat mammary tumors.

Inhibition by an extract of Ocimum sanctum of DNA-binding activity of 7,12-dimethylbenz[a]anthracene in rat hepatocytes in vitro.

Ocimum sanctum is a traditional medicinal plant. Previous studies have shown that extracts of O. sanctum inhibit the induction of skin papillomas in mice by 7,12-dimethylbenz[a]anthracene (DMBA). In the present study, primary cultures of rat hepatocytes were treated with 0-500 microg of O. sanctum extract for 24 h and then with DMBA (10 or 50 microg) for 18 h. Cells were then harvested and their DNA was isolated and analyzed by 32P-postlabelling. A significant reduction in the levels of DMBA-DNA adducts was observed in all cultures pretreated with O. sanctum extract. This effect was more pronounced at the lower dose of DMBA (10 microg). Hepatocytes which were treated with the highest dose of extract (500 microg) showed a maximum reduction of 93% in the mean values of DMBA-DNA adducts. The viability of the cells was not adversely affected by pretreatment with extract. Our findings suggest that O. sanctum leaf extract blocks or suppresses the events associated with chemical carcinogenesis by inhibiting metabolic activation of the carcinogen.

Modulation of phase I and phase II xenobiotic-metabolizing enzymes by selenium-enriched garlic in rats.

Previous research showed that treatment with selenium-enriched garlic (Se-garlic) was able to inhibit the initiation phase of mammary carcinogenesis in the dimethyl-benz[a]anthracene (DMBA) model in rats. The present study was designed to investigate the following parameters: 1) DMBA-DNA adduct formation in liver and mammary gland, 2) urinary excretion of DMBA metabolites, 3) phase I and phase II xenobiotic-metabolizing enzymes, and 4) tissue selenium levels as a function of Se-garlic supplementation. Prior feeding with an Se-garlic-containing diet (at 3 ppm Se) for two weeks resulted in a consistent reduction of all DMBA adducts in liver and mammary gland. This was accompanied by a 40% increase in urinary excretion of DMBA metabolites over a two-day period. Several liver P-450 enzymes were examined in rats fed a diet supplemented with 1, 2, or 3 ppm Se. Compared with controls receiving 0.1 ppm Se, no significant alteration in activity was detected with respect to P-450 1A1 (responsible for DMBA activation), 1A2, 2B1, 2E1, and 3A4. In contrast, glutathione S-transferase and uridine 5'-diphosphate-glucuronyltransferase activities were elevated to a maximum of 2- to 2.5-fold in liver and kidney. As expected, there was a dose-dependent elevation of selenium concentrations in liver, kidney, mammary gland, and plasma as a function of the level of Se-garlic supplementation. Our data seem to suggest that an increased detoxification of carcinogen via the phase II conjugating enzymes might represent a mechanism of tumor suppression by Se-garlic.

Chemoprevention of mammary cancer by diallyl selenide, a novel organoselenium compound.

Previous research has demonstrated that structurally distinctive organoselenium compounds are superior to the corresponding sulfur analogs in cancer prevention. The present study was designed to extend this observation to diallyl selenide (DASe), a volatile synthetic compound, and diallyl sulfide (DAS), a flavor component of garlic. Their anticarcinogenic activities were evaluated using the 7,12-dimethylbenz(a)-anthracene (DMBA)-induced mammary tumor model. Rats were gavaged three times with DASe (6 or 12 mumol/kg body wt) or DAS (300, 900 or 1,800 mumol/kg) at 96, 48 and 24 hours before DMBA treatment. Significant tumor inhibition was found with the two doses of DASe and the highest dose of DAS. Based on these results, DASe appears to be at least 300 times more active than DAS. Analysis of total DMBA-DNA binding and individual DNA adducts in the mammary gland and liver showed that DASe had no effect on these parameters, suggesting that DASe might influence some unknown risk-associated events other than carcinogen activation/detoxification. Although the mechanism of action of DASe remains to be elucidated, its potential relevance to natural products will be discussed in the context of the chemistry of selenium-enriched garlic which has been reported to be effective in cancer protection in several studies.

Dietary rosemary suppresses 7,12-dimethylbenz(a)anthracene binding to rat mammary cell DNA.

Commercially available ground rosemary powder was examined for its ability to modify the in vivo binding of 7,12-dimethylbenz(a)anthracene (DMBA) metabolites to mammary cell DNA in 55-d-old rats fed diets containing varying quantities and types of lipids. Supplementing a casein-based diet containing 20% corn oil with 1 % rosemary for 2 wk reduced by 76% the occurrence of DMBA-induced DNA adducts occurring 24 h after treatment with 50 mg DMBA/kg body weight. A comparable reduction in DNA adducts (66%) occurred when 0.5% rosemary was added to a diet containing 20% corn oil, and the quantity of DMBA given was reduced to 25 mg/kg body weight. The reduction in the occurrence of adducts occurring 24 h after DMBA treatment caused by 0.5% dietary rosemary was greater (P < 0.05) when added to a diet containing 20% corn oil than when added to a diet containing 5% corn oil and 15% coconut oil. Rosemary, 1% but not 0.5%, reduced DMBA-induced DNA adducts when the diet contained 5% corn oil. These studies demonstrate that rosemary is effective in reducing the binding of DMBA metabolites to rat mammary cell DNA. Furthermore, the present studies demonstrate that the benefits of rosemary are dependent on the source and concentration of dietary lipids.

Conjugated linoleic acid suppresses mammary carcinogenesis and proliferative activity of the mammary gland in the rat.

Conjugated linoleic acid (CLA) is a collective term which refers to a mixture of positional and geometric isomers of linoleic acid. It is naturally occurring in meat and dairy products. We have previously reported (Ip, C., Chin, S. F., Scimeca, J. A., and Pariza, M. W. Cancer Res., 51: 6118-6124, 1991) that 1% CLA in the diet suppressed mammary carcinogenesis in rats given a high dose (10 mg) of 7,12-dimethylbenz(a)anthracene. In the present study, dietary CLA between 0.05 and 0.5% was found to produce a dose-dependent inhibition in mammary tumor yield when fed chronically to rats treated with a lower dose (5 mg) of 7,12-dimethylbenz(a)anthracene. Short-term CLA feeding for 5 weeks, from weaning to the time of carcinogen administration at 50 days of age, also offered significant protection against subsequent tumor occurrence. This period corresponds to maturation of the mammary gland to the adult stage in the rat. The inhibitory response to short-term CLA exposure was observed with the use of 2 different carcinogens: 7,12-dimethylbenz(a)anthracene and methylnitrosourea. The fact that CLA was protective in the methylnitrosourea model suggests that it may have a direct modulating effect on susceptibility of the target organ to neoplastic transformation. The proliferative activity of the mammary epithelium was assessed by the incorporation of bromodeoxyuridine. Immunohistochemical staining results showed that CLA reduced the labeling index of the lobuloalveolar compartment, but not that of the ductal compartment of the mammary tree. Since the lobuloalveolar structures are derived from the terminal end buds which are the sites of carcinogenic transformation, the above finding is consistent with the bioassay data of tumor inhibition. Thus, changes in gland development and morphogenesis may be a locus of action of CLA in modulating mammary carcinogenesis. CLA is a unique anticarcinogen because it is present in foods from animal sources. Furthermore, its efficacy in cancer protection is manifest at dietary concentrations which are close to the levels consumed by humans.

Dietary selenite modifies glutathione metabolism and 7,12-dimethylbenz(a)anthracene conjugation in rats.

The present studies determined the impact of dietary selenite on glutathione homeostasis in liver and mammary tissue and its relationship to biliary excretion of 7,12-dimethylbenz(a)anthracene (DMBA) conjugates. In Experiment 1, liver and mammary tissue concentration of reduced glutathione (GSH) and activities of gamma-glutamylcysteine synthetase (GCS), glutathione reductase (GR) and glutathione S-transferases (GST) were positively correlated with tissue selenium concentration in female rats fed semipurified diets supplemented with sodium selenite (0.05 to 4 mg Se/kg). The magnitude of the response was dependent upon total selenite intake and the tissue examined. Glutathione peroxidase activity did not correlate with tissue GSH concentration. Because both selenite and BHT have been reported to elevate liver GSH, Experiment 2 compared these agents (4 mg Se/kg and 6 g/kg BHT/kg, respectively) on the biliary excretion of DMBA metabolites. Five major biliary DMBA conjugates, three GSH and two beta-glucuronide, were identified. Dietary addition of selenite or BHT enhanced the excretion of these DMBA conjugates by over 100% during the 15-h collection period. These investigations suggest that dietary selenium can alter the concentration of GSH and the activities of three glutathione-dependent enzymes in mammary and liver, accounting for part of the expanded biliary excretion of DMBA conjugates. Enhanced biliary loss of DMBA conjugates likely relates to the reported depression in DMBA binding to mammary cell DNA and the inhibition of DMBA carcinogenesis caused by dietary selenite.

Impact of various sources of garlic and their constituents on 7,12-dimethylbenz[a]anthracene binding to mammary cell DNA.

The present studies assessed the impact of various sources of garlic and their constituents (water- and ethanol-extracts and S-allylcysteine) on the in vivo binding of the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) to rat mammary cell DNA. The provision of dietary raw garlic powder (2%) or its water-extract (1.5%) reduced DMBA-DNA binding by 33 and 46% respectively. Dietary supplementation with a commercially available deodorized garlic powder (powder A) at 2 or 4% depressed the occurrence of adducts by 50 and 78% respectively, while providing a commercially available high sulfur garlic preparation (powder B) at 2% reduced binding by 56%. A pair-feeding study revealed that the depression in carcinogen binding was independent of food intake or weight gain. Although 1% raw garlic powder did not significantly influence the occurrence of DMBA-DNA adducts, an equivalent as the water-extract (0.75%), the ethanol-extract (0.015%) or commercially available powders (A and B) reduced DMBA adducts in mammary tissue by 44, 25, 71 and 65% respectively. Dietary fortification with S-allylcysteine (SAC), a water-soluble constituent of processed garlic, caused a progressive decrease in the binding of DMBA to DNA. Studies with SAC suggest the primary effect of garlic and its constituents is on the bioactivation and binding of the carcinogen rather than DNA repair. These data reveal that several forms of garlic are effective, although variable, in altering carcinogen bioactivation and presumably chemically induced carcinogenesis.

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.

Age, dietary selenium and quantity of 7,12-dimethylbenz(a)anthracene influence the in vivo occurrence of rat mammary DNA adducts.

The present studies determined the impact of age, dietary selenium and 7,12-dimethylbenz(a)anthracene (DMBA) dosage on the occurrence of DMBA-DNA adducts in rat mammary tissue. Diets formulated to contain selenium, as sodium selenite, at 0.1 (control) or 2.0 mg/kg were fed for 2 wk before DMBA treatment. Food intake and weight gain were not influenced by selenium intake. Anti- and syn-dihydrodiol epoxide adducts reached maximum binding by 24 and 36 h, respectively, after treatment with DMBA. Consumption of the diet containing 2.0 mg Se/kg inhibited the appearance of both anti- and syn-adducts by approximately 50% compared with controls. The occurrence of DMBA-DNA adducts correlated with a dosage of DMBA from 5 to 50 mg/kg body wt (r greater than or equal to 0.95). The ability of supplemental selenite to lower DMBA binding to mammary cell DNA increased as the quantity of the carcinogen administered increased. DMBA-DNA binding was found to increase with the increasing age of the rat. Nevertheless, dietary selenium supplementation was effective in reducing DMBA binding to DNA in all ages examined. These data confirmed the ability of dietary selenium to inhibit the in vivo metabolism of DMBA under a variety of conditions.