DMBA Treatment Research Articles

Prepubertal exposure to zearalenone or genistein reduces mammary tumorigenesis.

Prepubertal exposure to a pharmacological dose (500 mg kg−1) of the phyto-oestrogen genistein can reduce the incidence and multiplicity of carcinogen-induced mammary tumours in rats. However, such an exposure also disrupts the function of the hypothalamic–pituitary–gonadal axis, making it unsuitable for breast cancer prevention. We studied whether prepubertal exposure to genistein at a total body dose broadly comparable to the level typical of Oriental countries, approximately 1 mg kg−1 body weight, affects mammary tumorigenesis. We also studied whether prepubertal exposure to zearalenone, a major source for phyto-oestrogens in the USA, influences breast cancer risk. Prepubertal rats were treated between postnatal days 7 and 20, with 20 μg (~ 1 mg kg−1 body weight) of either genistein or zearalenone. Zearalenone exposure significantly reduced both the incidence and multiplicity of mammary tumours induced by 7,12-dimethylbenz(a)anthracene (DMBA). Genistein exposure significantly reduced tumour multiplicity, but not tumour incidence, when compared with vehicle-treated animals. Furthermore, 60% of the tumours in the genistein group were not malignant, while all the tumours analysed for histopathology in the vehicle and zearalenone groups were adenocarcinomas. A higher number of differentiated alveolar buds, and lower number of terminal ducts, were present in the DMBA-treated mammary glands of the phyto-oestrogen exposed rats. The concentration of oestrogen receptor (ER) binding sites after the DMBA treatment was low in the mammary glands of all groups but a significantly higher proportion of the glands in the zearalenone exposed rats were ER-positive (i.e. ER levels ≥ 5 fmol mg−1 protein) than the glands of the vehicle controls. Our data suggest that a prepubertal exposure to a low dose of either zearalenone or genistein may protect the mammary gland from carcinogen-induced malignant transformation, possibly by increasing differentiation of the mammary epithelial tree. © 1999 Cancer Research Campaign

Heating Garlic Inhibits Its Ability to Suppress 7,12-Dimethylbenz(a)anthracene-Induced DNA Adduct Formation in Rat Mammary Tissue1–4

The present studies compared the impact of heating, either by microwave or convection oven, on the ability of garlic to reduce the in vivo bioactivation of 7,12-dimethylbenz(a)anthracene (DMBA) in 55-d-old female Sprague-Dawley rats. In study 1, rats were fed a semipurified casein-based diet and treated by gastric gavage thrice weekly for 2-wk with crushed garlic (0.7 g in 2 mL corn oil) or the carrier prior to DMBA treatment (50 mg/kg body weight). Providing crushed garlic reduced by 64% (P < 0.05) the quantity DMBA-induced DNA adducts present in mammary epithelial cells compared to controls. In study 2, microwave treatment for 60 s, but not 30 s, decreased (P < 0.05) the protection provided by garlic against DMBA-induced adduct formation. In study 3, allowing crushed garlic to stand for 10 min prior to microwave heating for 60 s significantly (P < 0.05) restored its anticarcinogenic activity. Microwave heating of garlic for 30 s resulted in a 90% loss of alliinase activity. Heating in a convection oven (study 4) also completely blocked the ability of uncrushed garlic to retard DMBA bioactivation. Study 5 revealed that providing either 0.105 μmol diallyl disulfide or S-allyl cysteine by gastric gavage thrice weekly for 2 wk was effective in retarding DMBA bioactivation but isomolar alliin was not. These studies provide evidence that alliinase may be important for the formation of allyl sulfur compounds that contribute to a depression in DMBA metabolism and bioactivation.

A Preliminary Study of the Effect of Plantago Ovata Forsk on the Development of 7, 12-Dimethylbenz[a]anthracene-initiated Rat Mammary Tumors under the Influence of Hypercholesterolemia

As a preliminary study to elucidate whether Plantago ovata forsk (PO) exhibits inhibiting effects on mammary carcinogenesis under a condition of hypercholesterolemia, 50 female Sprague-Dawley rats were first given a single intragastric dose of 200 mg/kg body weight of 7, 12-dimethylbenz[a]anthracene (DMBA). Ten of 50 rats died of acute toxicity of DMBA within one week. From one week later, the survived animals were divided into 4 groups: 13 rats in group 1 and 7 in group 2 received high cholesterol diets (HC) with and without 5% PO supplementation for 26 weeks, respectively. Eleven rats in group 3 and 9 in group 4 received basal diet (BD) with and without 5% PO supplementation for the same period, respectively. One to three rats in groups 1 to 4 died of mammary tumors, lymphomas and/or adrenal impairment attributable to DMBA treatment during the treatment period. Group 1 showed a tendency to decrease in the number of palpable mammary masses as compared with group 2, whereas group 3 showed a tendency to higher values compared with group 4. At the termination of the study, the serum levels of total cholesterol in group 1 were significantly lower than those in group 2 and the number of mammary masses was significantly decreased. Histopathologically, this decrease was mainly due to the decreased incidences of mammary adenocarcinomas, while no significant difference in the multiplicity of mammary tumor was observed between BD+PO and BD alone groups. The results of the present study suggest a possibility that PO exerts inhibiting effects on mammary carcinogenesis by decreasing circulating cholesterol levels in a rat two-stage mammary carcinogenesis model.

Spindle disturbances induced by benzo[ a]pyrene and 7,12-dimethylbenz[ a]anthracene in a Chinese hamster cell line (V79-MZ) and the stability of the numerical chromosome aberrations that follow

We previously reported that benzo[ a]pyrene (BP) and 7,12-dimethylbenz[ a]anthracene (DMBA) induce aneuploidy and polyploidy, respectively, in the Chinese hamster cell line V79-MZ in the absence of S9 mix. In the present study we investigated the effect of BP and DMBA on the mitotic spindle. BP caused incomplete spindle formation and DMBA inhibited spindle formation completely. The combined results indicate that incomplete spindles caused by BP resulted in aneuploidy, and the absence of spindle formation caused by DMBA resulted in polyploidy. The induced polyploidy was stable for several serial passages in fresh medium. BP and DMBA induced different chromosome number distributions. After BP treatment, the normal distribution of chromosome number was restored in 4 days. After DMBA treatment, on the other hand, a tetraploid peak was maintained for 2 months following an initial transient broad distribution of chromosome number after 1 day. The results suggest that different mechanisms were involved in the induction of numerical aberrations by BP and DMBA. Furthermore the induction of numerical aberrations by BP and DMBA was reproducible over 5 months of passages. In four clones tested, the frequency of cells with the modal chromosome number in control cultures gradually decreased from 82% on the average just after cloning to 62% 5 months later. BP and DMBA induced characteristic ploidy changes following succeeding cell passages for up to 5 months, indicating that the ability to respond to BP and DMBA was stable for that period of time. Because these findings were specific to V79-MZ cells, this cell line might be a good tool for studying chemicals that induce numerical chromosome aberrations.

Ultraviolet B–induced squamous epithelial and melanocytic cell changes in a xenograft model of cancer development in human skin

We previously demonstrated that precancers (actinic keratoses and dysplasias) and squamous cell carcinomas (SCCs) develop in one quarter of human neonatal foreskins grafted onto recombinase-activating gene-1-knockout mice treated once with 7,12-dimethylbenz[a]anthracene (DMBA) followed by chronic intermediate-range ultraviolet (UV) B light irradiation. The goals of this study were to determine if a longer UVB exposure followed by further observation would increase the number of precancers and invasive cancers and to evaluate whether this model results in changes in p53 expression and cell proliferation similar to those seen in sun-damaged normal skin, actinic keratoses, and SCCs. The treatment consisted of a single dose of DMBA followed by 500 J/m2 UVB radiation administered three times weekly for at least 5 mo. Histologic changes (cysts, hyperplasias, precancers, and/or invasive cancers) were seen in 24 of 25 treated xenografts but not in controls. Ten of 25 grafts (40%) had two or more histological changes, and two human SCCs developed. After seven or more months of UV exposure and a total time from DMBA treatment to killing of 12-18 mo, 83% (15 of 18) of specimens developed squamous precancer or SCC of human origin, and 44% (eight of 18) developed melanocytic hyperplasia or melanoma. The change from moderate dysplasias to SCC required longer UV exposure (median, 11 mo), and 5 mo more observation than did the development of mild dysplasias (median UV exposure, 7 mo; median DMBA to death time, 12 mo). There was a direct correlation between both p53 expression and cell proliferation and the degree of histologic alteration both in squamous epithelial and melanocytic cells.

Histologic characterization of rat ovarian carcinoma induced by intraovarian insertion of a 7,12-dimethylbenz[a]anthracene-coated suture: common epithelial tumors of the ovary in rats?

The surface epithelium of the human ovary simulates the mullerian form in tumor formation. In experimental animals, however, such a phenomenon has not previously been observed. A chemical carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA), was heated, and the central portion of a 3-0 silk suture was immersed in the melted carcinogen. After vaginal cytology, the DMBA-coated silk was inserted into the ovaries of 40 Wistar strain rats age 7 weeks. The animals were sacrificed when a tumor mass became large enough to extend the abdominal wall. The experimental period lasted for 60 weeks. The tumor histology was compared with that in human counterparts and the mullerian derivatives in rats. In 19 rats, including 4 animals with cornified vaginal smears at the time of DMBA treatment, an ovarian carcinoma developed within 36 weeks. In 17 epithelial tumors, the neoplastic cells proliferated to form papillary or glandular structures, and the lining epithelium consisted of flattened or cuboidal cells and columnar cells, often with pseudostratified nuclei. Adenocarcinoma components transformed into squamous cells in two cases. The remaining two neoplasms were pure mesenchymal tumors with histology of a fibrosarcomatous tumor, and one of them contained heterologous malignant osteoid components. In the remaining 21 rats, including 5 with cornified smears, no tumors developed during the experimental period. The histologies of DMBA-induced rat ovarian carcinoma simulated the epithelia of rat ovarian surface, fallopian tube, endometrium, and uterine cervix. The results of this study suggest that the surface epithelium of rat ovary also simulates the mullerian form in tumor formation.

Analysis ofras gene mutational spectra in epidermal papillomas from K6/ODC transgenic mice

In standard mouse strains, a high proportion (more than 90%) of epidermal tumors produced by initiation with 7,12-dimethylbenz[a]anthracene and promotion with a variety of chemical agents contain an activating mutation in codon 61 (A182-->T) of the c-Ha-ras gene. We analyzed the ras mutational spectra in 69 tumors induced by DMBA in a unique transgenic model, the K6/ODC mouse. In this model, low-dose DMBA treatment is sufficient per se for tumor induction, so tumor promotion with chemical agents is not required. In contrast to previous studies in standard mouse strains, our study showed that less than 50% of epidermal tumors from K6/ODC mice contained an activating codon 61 c-Ha-ras mutation (A182-->T). This result was obtained in mice initiated either as newborns (when the transgene is not expressed) or as adults (when the transgene is fully expressed). Analysis of other codon hot-spots and other ras genes revealed the presence of three codon 12 and 20 codon 61 (A182-->T) mutations in the c-Ki-ras gene in the 36 tumors that did not have c-Ha-ras mutations. We concluded that promotion in this model, by means of constitutive ornithine decarboxylase expression, causes the clonal expansion of a population of initiated cells not promoted by chemical agents.

Placental glutathione S-transferase isoenzyme expression in polycyclic aromatic hydrocarbon-induced hamster buccal pouch mucosa.

Placental glutathione S-transferase (GST-P) immunohistochemical activity in polycyclic aromatic hydrocarbon (PAH)-induced hamster buccal pouch mucosa (HBPM) was investigated. Equimolar concentrations of 3,4-benzo[a]pyrene (BP), benz[a]anthracene (BA) and 20-methylcholanthrene (MC) in mineral oil solution were applied three times per week for up to 20 weeks to bilateral pouches of the hamsters. Control pouches were treated with 7,12-dimethylbenz[a]anthracene (DMBA), turpentine and mineral oil, respectively. A significant increase in the number of GST-P foci in the dysplastic pouches was found when compared with the hyperkeratotic and hyperplastic pouches after 5 weeks' DMBA treatment. The average numbers of GST-P positive stainings were significantly higher in DMBA-treated groups than in BP-, BA- and MC-treated groups killed at 5, 20, 30 and 40 weeks. No GST-P positivity was found in any pouches treated with turpentine and mineral oil observed at various periods. These results highlight the carcinogen-specificity of GST-P chemically induced in HBPM and indicate that induction of GST-P in pouch mucosa is linked with the carcinogenic potency of PAHs. The present study underpins the hypothesis that GST-P chemically induced in HBPM is an early marker of ongoing squamous cell carcinogenesis.

Chemopreventive efficacy of curcumin-free aqueous turmeric extract in 7,12-dimethylbenz[ a]anthracene-induced rat mammary tumorigenesis

The modulating effects of turmeric (T), ethanolic turmeric extract (ETE) and curcumin-free aqueous turmeric extract (CFATE) on the initiation or post-initiation phases of DMBA-induced mammary tumorigenesis were investigated in female Sprague–Dawley rats. Dietary administration of 1% T/0.05% ETE 2 weeks before, on the day of DMBA treatment (day 55) and 2 weeks after the single dose (15 mg/animal) of DMBA (during the initiation period) resulted in significant suppression of DMBA-induced mammary tumorigenesis as seen by a reduction in tumor multiplicity, tumor burden and tumor incidence. However, simultaneous administration of 1% T-derived CFATE as the sole source of drinking water during the initiation phase did not suppress DMBA-induced mammary tumorigenesis. Dietary administration of 1% T/0.05% ETE or 1% T-derived CFATE as the sole source of drinking water starting 48 h after DMBA treatment and continuing until the end of the experiment (during the post-initiation period) resulted in significant suppression of DMBA-induced mammary tumorigenesis as seen by reduction in the tumor multiplicity and/or tumor burden although tumor incidence was unaffected. The present data clearly indicate that dietary administration of T/ETE showed strong chemopreventive activity during initiation as well as post-initiation phases of DMBA-induced rat mammary tumorigenesis while CFATE was found to be weakly active only when it was administered during the post-initiation phase.

Intrasplenic grafting of ovarian tissue containing 7, 12-dimethylbenz[a]anthracene.

To produce malignant granulosa cell tumors, ovarian tissue containing 7,12-dimethylbenz[a]anthracene (DMBA) was autografted in the spleen of 20 castrated rats. Adenocarcinoma developed in 8 rats within 6 months from the transplantation. Granulosa cell tumor arose from the intrasplenic graft, in 6 rats at the 14th month. The neoplastic cells were diffusely arranged in sheet showing focal luteinization. In 3 cases extrasplenic spreads were found on the peritoneal surface. Since malignant ovarian tumors seldom develop by intrasplenic grafting alone, the DMBA treatment may contribute to the occurrence of malignant granulosa cell tumor.

Verification in syngeneic hamsters of in vitro transformation of hamster oral mucosa by 7,12-dimethylbenz(a)anthracene

The carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) has been used to induce oral carcinogenesis of the hamster buccal mucosa in an experimental model that exhibits many of the genetic, biochemical and pathological features of human oral squamous cell carcinoma. To complement this in vivo process we have established an in vitro transformation procedure that involved the treatment of normal hamster oral mucosal keratinocytes (NHKs) with DMBA. Uptake of DMBA in NHKs was verified by observing autofluorescence of DMBA in the oral mucosal cells. Treatment doses ranged from 5, 50 and 200 ng and the NHKs were generally treated with DMBA for 1-14 days. The 200 ng dose proved to be toxic to these cells. The 5 and 50 ng treatments were found to maintain the viability of the NHKs and demonstrate anchorage-independent agarose growth, producing 18 and 40 colonies, respectively, after 14 days of treatment. Characterisation assays included determinations for cellular growth through plating efficiency, counting of cell colony number, and 3H-thymidine incorporation. Differentiation was ascertained by counting cornified cells, specification of either high or low molecular weight keratins, the percentage of cells expressing gamma glutamyl-transpeptidase (GGT), the level of p53 expression, and a determination of cell cycle. After 24 h the plating efficiency of the NHKs was found to be slightly increased following treatment with a 5 or 50 ng dose of DMBA compared to the untreated NHKs. After 14 days of incubation these doses also enhanced the number of colonies formed by the NHKs (e.g. plating efficiency). In contrast, the number of cornified cells was reduced in these colonies, while immunohistochemistry disclosed an increase in the number of NHKs expressing low molecular weight keratins, significantly lower levels of high molecular weight keratins and high levels for GGT. Flow cytometric analysis verified an increase in p53 expression (e.g. p53 wild type, 19% and p53 mutant, 66%). Cell cycle analysis of NHKs treated with DMBA (5 ng) demonstrated a shift in the number of cells in S phase (17.2%) and G2 + mitosis (11.0%). Cells from this DMBA treatment group were injected into syngeneic hamster recipient buccal pouches (10 x 10(6) cells/0.25 ml). Squamous carcinomas grew in four of six hamster buccal pouches as determined by histopathological analysis. The in vitro assay system will enhance our ability to define the genetic and molecular changes related to chemical carcinogenesis and oral malignant transformation.

Tissue distribution of DNA adducts in rats treated by intramammillary injection with dibenzo[ a,l]pyrene, 7,12-dimethylbenz[ a]anthracene and benzo[ a]pyrene

Dibenzo[ a,l]pyrene (DBP) has recently emerged as a potent environmental carcinogen having greater carcinogenicity in the rat mammary epithelial glands than 7,12-dimethylbenz[ a]anthracene (DMBA), previously considered to be the most potent mammary carcinogen and benzo[ a]pyrene (BP), a ubiquitous environmental carcinogen. Previous studies on the tumor-initiating potential of DBP, DMBA, and BP demonstrated that DBP was 2.5 times more potent in inducing the tumors in mouse skin and rat mammary glands than DMBA; BP was a weak mammary carcinogen in these animals. The present study was designed to investigate if the significantly increased mammary carcinogenicity of DBP over DMBA and BP was related to increased DNA adduction at the target site. Female Sprague–Dawley rats were treated by intramammillary injection with an equimolar dose of 0.25 μmol/gland of DBP, DMBA, and BP at the 3rd, 4th and 5th mammary glands on both sides. 32P-Postlabeling analysis of mammary epithelial DNA of rats treated with DBP produced two major (nos. 3 and 6) and at least 5 minor adducts. DMBA treatment resulted in one major and 4 minor DNA adducts while BP produced one major and two minor adducts. Quantitation of the adduct radioactivity revealed that DNA adduction was 6- and 9-fold greater in DBP-treated animals than in BP- and DMBA-treated animals, respectively. The adduct levels per 10 9 nucleotides in mammary epithelial cells for DBP, BP and DMBA were in the following descending order: 1828±378, 300±45 and 207±72, respectively. Tissue distribution of DNA adducts in non-target organs following DBP treatment showed similar adduct pattern as found in the mammary epithelial cells except the liver, which resulted in 4 additional adduct spots; vehicle-treated tissue DNA processed in parallel did not show any detectable adducts. DMBA- and BP–DNA adduct patterns in various tissues were similar to that found in mammary epithelial cells, however, significant quantitative differences were found; BP–DNA adducts were undetectable in the pancreas and bladder. Quantitation of adduct radioactivity showed a 15- to 60-fold lower DBP-DNA adduction in these tissues than the levels found in the mammary tissue; similarly 5–20 and 30–100 times lower DNA adduction was found following treatment with DMBA and BP, respectively. The significantly increased binding of DBP to the mammary epithelial DNA over BP and DMBA is in concordance with its known higher mutagenicity and tumorigenicity.

Post-initiation inhibitory effects of green tea catechins on 7,12-dimethylbenz[a]anthracene-induced mammary gland carcinogenesis in female Sprague-Dawley rats.

The dose-dependence of green tea catechin (GTC) effects on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary gland carcinogenesis were investigated in female Sprague-Dawley rats. Groups of 20 6-week-old rats were treated with dietary 1, 0.1 or 0.01% GTC for 2 weeks and then basal diet alone for 35 weeks. At the end of week 1, they received a 25 mg/kg body weight intragastric dose of DMBA. Further groups of 20 7-week-old rats each were given an intragastric dose of 25 mg/kg body weight DMBA, and starting 1 week after DMBA treatment they were placed on diet containing 1, 0.1 or 0.01% GTC or basal diet alone for 35 weeks. Control rats were given 1% GTC or basal diet alone. The final incidences and multiplicities of mammary tumors were not significantly different between the groups treated with GTC at the same time as DMBA, compared to the DMBA alone control group. On the other hand, the final multiplicities of mammary tumors in groups treated with 1% GTC (P < 0.05) or 0.01% GTC (P < 0.01), but not 0.1% GTC, after DMBA treatment were significantly decreased as compared to the control value. These results indicate that whereas GTC may inhibit mammary carcinogenesis in the post-initiation stage, the effect is weak and not dose-dependent.

Inhibitory effects of dietary protocatechuic acid and costunolide on 7,12-dimethylbenz[a]anthracene-induced hamster cheek pouch carcinogenesis.

The modifying effects of dietary exposure to two natural products, protocatechuic acid (PCA) and Costunolide during the development of neoplasms in oral carcinogenesis initiated with 7,12‐dimethyl‐benz[a]anthracene (DMBA) were investigated in male Syrian golden hamsters. All hamsters except those in the test chemical alone and control groups received DMBA (0.5%) in mineral oil to the right buccal pouch 3 times per week for 4 or 6 weeks. At 13 weeks of age, the groups exposed to DMBA were fed diet containing PCA or Costunolide at a dose of 0.2 g/kg diet (200 ppm) for 17 weeks. The other groups consisted of hamsters given mineral oil alone for 6 weeks, or given 200 ppm PCA or Costunolide alone, or untreated. All animals were necropsied at the termination of the experiment (week 24). PCA or costunolide significantly decreased the tumor burden (P<0.001‐P<0.05) and the extent of dysplastic areas (%) (P<0.001‐P<0.05). PCA significantly decreased the mean number of AgNORs/nucleus (P<0.05). The BrdUrd‐labeling index was reduced by dietary administration of test compounds, though not significantly. These results suggest that PCA and costunolide inhibited hamster buccal pouch carcinogenesis and such inhibition may be related to suppression of cell proliferation in the buccal mucosa. It was also found that telomerase activity expressed in neoplastic and preneoplastic lesions of hamster buccal pouch epithelium after DMBA treatment correlated with the histopathological degree of malignancy.

Protective effect of leuprolide acetate on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis in rats

The anti-carcinogenic effect of TAP-144-SR biodegradable microcapsules of copoly (DL-lactic/glycolic acid) copolymer containing a potent LHRH agonist, TAP-144 (D-Leu6-(des-Gly10-NH2)-LHRH ethylamide, leuprolide acetate) was investigated in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumours. At 50 days of age, all rats were given 20 mg doses of DMBA by gastric intubation. Rats on the Initiation Group were given a single s.c. injection of TAP-144-SR (3 mg/kg) 1 week before DMBA treatment, and rats in the Promotion Group were given repeated s.c. injections every fourth week from 1 week after DMBA treatment until the end of the experiment. In the Initiation Group, tumour incidence and multiplicity were reduced to 20.8% and 15.0% of the Control Group values, respectively. In the Promotion Group, tumour incidence and multiplicity were also reduced to 17.7% and 7.9%, respectively. It was suggested that TAP-144-SR may be a potential candidate as an agent for hormonal chemoprevention in breast cancer.

Impact of dietary fatty acids on 7,12-dimethylbenz[a]anthracene-induced mammary DNA adducts.

These studies examined the impact of dietary corn oil and its major constituent fatty acids on the occurrence of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary DNA adducts. In study 1, rats were fed diets containing 5, 10 or 20% corn oil for 2 weeks prior to DMBA treatment (25 mg/kg). Mammary DNA adducts increased significantly (P < 0.05) as the quantity of dietary corn oil increased. Liver glutathione S-transferase (GST) activities increased while UDP-glucuronosyltransferase (UGT) activities decreased as the quantity of dietary corn oil increased. Increased adducts resulting from greater corn oil consumption were positively correlated with GST and negatively correlated with UGT activities. In study 2, rats were fed diets containing 5 or 20% corn oil, or 5% corn oil supplemented with 1.67% palmitic, 3.81% oleic or 8.78% linoleic acid (quantities in 15% corn oil) for 2 weeks prior to DMBA treatment (50 mg/kg). Total mammary DNA adducts were 75, 136 and 156% greater in rats fed the 20% corn oil, oleic acid-supplemented and linoleic acid-supplemented diets, respectively, than in those fed the 5% corn oil diet. Palmitic acid supplementation did not affect the occurrence of adducts. Adducts in study 2 did not correlate with GST or UGT activities. These studies demonstrate that enhanced DMBA bioactivation caused by increased corn oil consumption can be at least partially explained by increased intake of oleic and linoleic acids.

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.

Dietary Components Modify the Ability of Garlic to Suppress 7,12-Dimethylbenz(a)anthracene-Induced Mammary DNA Adducts

Various dietary components were evaluated as factors influencing garlic's ability to depress rat mammary cell DNA adducts resulting from 7,12-dimethylbenz(a)anthracene (DMBA) treatment. Diets with or without garlic powder (20 g/kg) were provided for 2 wk before DMBA treatment (25 mg/kg body weight). Rats fed diets containing 36 g casein/100 g diet had 31% fewer (P < 0.05) mammary cell DNA adducts than those fed 12g/100 g. Garlic supplementation significantly (P < 0.05) reduced DNA adducts in rats fed either 12 or 36 g casein/100 g by 35 and 32%, respectively. In the absence of dietary garlic, DNA adducts were 23% lower (P < 0.05) in rats provided a diet containing supplemental L-methionine at 0.9 g/100 g than at 0.3 g/100 g. However, adduct inhibition by garlic supplementation was greater in rats fed the lower (P < 0.05) amount of methionine (54 vs. 26% inhibition). Adduct levels in rats fed diets with 20 g corn oil/100 g were twice those occurring in rats fed 5 g/100 g (P < 0.05), regardless of adjustment for energy density. Garlic supplementation prevented the increase in DNA adducts caused by increasing dietary corn oil. Combining dietary supplements of garlic, selenite (0.5 mg/kg diet) and retinyl acetate (328 mg/kg diet) inhibited the occurrence of DNA adducts to a greater degree than when each was supplied individually. These studies demonstrate that while dietary garlic can reduce DNA adduct formation in mammary tissue caused by DMBA, this protection is influenced by several dietary components.

Sequential expression of myc-, ras-, oncogene products and EGF receptor during DMBA-induced tongue carcinogenesis

The mechanism leading to development of oral cancer has not been completely understood. It is currently believed that alternation of a number of genes can result in the development of epidermoid carcinomas. In this investigation, we used a 9,10-dimethyl 1,2-benzanthracene (DMBA)-induced carcinogenesis in a hamster tongue model to investigate the expression of c-myc, c-Ha-ms proteins and epidermal growth factor receptor (EGFr). During the DMBA-carcinogenesis of the tongue, the number of c-myc protein positive cells were increased in epithelial dysplasia and elevated throughout the process of tumorigenesis. The expression of c-Ha-ras protein was detected in normal epithelium. The level of c-Ha-ras protein expression was decreased in the dysplastic stage, and it was almost negative in squamous cell carcinomas. Detection of EGFr overexpression occurred only after 1-4 weeks of DMBA treatment, at a very early stage of tumor development, and increased through carcinogenesis varying individually within the malignant tissues. These results suggest that c-myc protein and c-Ha-ras protein expression may have an important role in malignant transformation, and the overexpression of EGFr can be correlated to very early stages of tumor development in the DMBA-induced in vivo tongue carcinogenesis.

Exposure of DMBA-treated female rats in a 50-Hz, 50 microTesla magnetic field: effects on mammary tumor growth, melatonin levels, and T lymphocyte activation.

There is growing public concern about the possible health risks, particularly increased cancer risks of exposure to magnetic fields (MF) associated with power distribution systems. Recently, we have started a series of animal studies to investigate this issue, using the DMBA (7,12-dimethylbenz[a]anthracene) model of breast cancer in female rats. In the present study, female rats were chronically exposed to a 50-Hz, 50 microTesla (microT) MF with or without DMBA treatment. Because alterations in circulating levels of the pineal hormone melatonin and impaired immune system functions have been involved in breast cancer growth, and both melatonin and immune system are thought to be targets for MF-effects, serum melatonin and the proliferative capacity of splenic lymphocytes were determined in MF-exposed and sham-exposed rats. For this purpose, 216 female Sprague-Dawley rats were divided into four groups. Two of the groups (with 99 animals each) received oral applications of DMBA and were either sham-exposed or exposed in a 50-Hz, 50 microT MF for 24 h/day 7 days/week for a period of 91 days. The other two groups (9 animals each) were either sham-exposed or MF-exposed without DMBA treatment. The exposure chambers and all other environmental factors were identical for MF-exposed and sham-exposed animals. The DMBA-treated animals were palpated once weekly to assess the development of mammary tumors. At the end of the three-month period of MF exposure, the number and size of mammary tumors was determined by autopsy. In controls, DMBA induced tumors in approximately 55% of the animals within the 3 month period of sham-exposure. Already 8 weeks after DMBA application, the MF-exposed group exhibited significantly more tumors than sham-exposed animals. At time of autopsy, significantly more MF-exposed DMBA-treated rats exhibited macroscopically visible mammary tumors than DMBA-treated controls, thus indicating that MF exposure enhances the development and growth of cancers in this model. Comparison of the data from 50 microT with recent data from other flux densities indicated that long-term MF exposure of DMBA-treated rats increases the incidence of palpable and/or macroscopically visible mammary tumors in a highly dose-related fashion. Determination of nocturnal serum melatonin after 9 and 12 weeks of exposure at 50 microT did not yield significant differences between MF-exposed rats and sham-exposed controls, whereas a marked suppression of T cell proliferative capacity was seen in MF exposed rats. The data add further evidence to the hypothesis that hormone-dependent tissues such as breast might be particularly sensitive to MF-effects and indicate that immune system depression is involved in the increased breast cancer growth observed in MF exposed rats.