Structure of 7,12-dimethylbenz(a)anthracene-guanosine adducts

Arene oxides have been proposed as the reactive intermediates in the process of carcinogenesis induced by polycyclic aromatic hydrocarbons. The present study defines the structures of four guanosine adducts formed by the reaction of 7,12-dimethylbenz[a]anthracene-5,6-oxide with polyguanylic acid. The modified polymer was hydrolyzed to nucleotides and the hydrophobic guanosine adducts separated from unmodified guanosine by LH-20 column chromatograhy. The adducts were further resolved into four components (I-IV) by reverse phase high pressure liquid chromatography. Analysis of the ultraviolet, circular dichroism, mass, and proton magnetic resonance spectra of these compounds, or their acetate and free base derivatives, indicates that in all four compounds the aromatic hydrocarbon is present on the 2 amino group of guanine. Compounds I and IV, and II and III constitute diastereoisomeric pairs, respectively. In the I and IV pair, the adducts result from addition at the 6 position of the original dimethylbenz[a]anthracene oxide, whereas in the II and III pair, the addition occurs at the 5 position. Indirect evidence suggests that trans opening of the oxide occurred in all cases but this remains to be established.     

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.     

Effect of metabolites of 7,12-dimethylbenz(a)anthracene on the dynamics of its fluorescence in the skin of hairless mice

The influence of two metabolites of 7,12-dimethylbenz(a)anthracene (DMBA)--7-hydromethyl-12-methylbenz(a)anthracene and 7,12-dihydroxymethylbenz(a)anthracene--on the dynamics of DMBA fluorescence was studied in the skin of mice. The first of the metabolites did not affect the dynamics of DMBA fluorescence, whereas the second one, when used in equimolar concentrations with DMBA, prolonged the duration of DMBA fluorescence in the skin. The same effect was observed in case of 7,8-benzoflavone, and inhibitor of DMBA metabolism.     

Cerebellar dysgenesis in rats by diaplacental effects of 7,12-dimethylbenz[a]anthracene

7,12-Dimethylbenz[a]anthracene (DMBA) administered iv to pregnant Sparague-Dawley rats produced cerebellar malfunction in at least 10% of the offspring. The underlying morphologic basis of the cerebellar symptomatology was found in maldevelopment of cerebellar cortex, ranging from focal loss of granule and Purkinje's cell layers to extensive areas of cortical disorganization with losses of granule neurons. An interference with the proliferation of the granule cell-layer primordium was suggested as a mechanism of this cerebellar dysgenesis. After a 200-day observation, no tumors were found in the offspring of the DMBA-treated, pregnant rats.     

Oestradiol and progesterone receptors in the mammary tissue and tumours of female Holtzman rats following 7,12-dimethylbenz(a)anthracene administration

A single dose of the carcinogen 7,12-dimethylbenz(a)-anthracene (DMBA) when administered to 55-day-old female Holtzman rats did not produce any change in the pattern or amount of oestradiol receptors in the breast tissues, when compared to age-matched control animals. The period of observation extended up to 280 days of age. Following DMBA administration to 102 animals, only 60% developed carcinomas of the breast, of which 59 and 50% were ER- an PgR-positive, respectively. This lower yield of steroid receptor-positive tumours in this strain of rats was also reflected in a very low incidence of regression of tumours to ovariectomy. Plausible explanations for both these findings have been offered.     

Evidence that iron-overload promotes 7,12-dimethylbenz(a)anthracene- induced skin tumorigenesis in mice

Iron overload is known to occur in West European and American populations due to the consumption of an iron-rich diet. There are also genetic disorders which lead to body iron overload. It has been shown that iron overload predisposes humans to an increased risk of cancer. In experimental animals, iron overload is known to enhance intestinal, colon, hepatic, pulmonary and mammary carcinogenesis. However, the mechanism by which iron overload enhances chemically-induced carcinogenesis is not known. In this study, we show that iron overload acts as a mild tumor promoter in mouse skin. Female albino swiss mice were given 1 mg iron/mouse parenterally for 2 weeks to induce iron overload. These animals showed a three-fold increase in cutaneous iron concentration as compared to normal mice. Tumors were initiated by topically applying 7,12-dimethylbenz(a)anthracene (DMBA). Appearance of the first tumor (latency period), percent tumor incidence and number of tumors/mouse were recorded. When compared to the control group, iron overload mice showed an increased incidence of tumors, from 25%-55% by week 20, and tumors appeared 4 weeks earlier. The number of tumors per mouse was four-fold higher in the iron overload group. The induction of cutaneous ornithine decarboxylase (ODC) activity and [3H]thymidine incorporation in cutaneous DNA were higher in iron overload groups as compared to normal control animals. Similar to other oxidant tumor promoters, iron overload enhanced cutaneous lipid peroxidation and xanthine oxidase activity and decreased catalase activity. Our results indicate that iron overload exerts a mild tumor promoting activity in mouse skin. Our data also show that oxidative stress generated by iron overload plays an important role in the augmentation of cutaneous tumorigenesis. These data may also have implications for the enhanced risk of cancer-induction following UVB exposure of human populations with iron overload.     

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 12 g/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.     

Inhibitory effect of a steroidal antiestrogen (EM-170) on estrone-stimulated growth of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat

Rats were trained on a successive delayed conditional discrimination task measuring memory for magnitude of reinforcement. In the study phase of the task, the rats were given one of two cereals. One cereal contained 25% sugar; the other 50% sugar. One of the two cereals was always designated the positive stimulus and the other the negative stimulus. This study phase was followed by the test phase in which the rat was shown an object which covered a food well. If the rat was given the negative stimulus in the study phase of the trial, no food reward was placed beneath the object. Whenever the positive stimulus was presented a food reward was available beneath the object. Performance was measured as the latency to uncover the food well. After reaching criterion level, the rats were given amygdala, hippocampal, or control lesions. Amygdala-lesioned rats showed significant deficits in performance, whereas no long-term deficits were observed for the hippocampal-lesioned groups even at longer retention delays. In additional experiments, it was shown that amygdala-lesioned, like normal, rats had similar taste preferences. Finally, normal and hippocampal-, but not amygdala-, lesioned rats transferred readily to different cereals containing 25% or 50% sugar. Thus, it appears that the amygdala, but not the hippocampus, plays a significant role in explicit data-based or working memory for affect information based on magnitude of reinforcement.     

Combination treatment with cisplatin and schizophyllan for 7,12-dimethylbenz(a)anthracene-induced rat ovarian adenocarcinoma

OBJECTIVE: Experimental chemotherapy was conducted to investigate the combined effect of Schizophyllan (SPG) and Cisplatin (CDDP). METHODS: Rats with 7,12-dimethylbenz(a)anthracene (DMBA)-induced ovarian adenocarcinoma received SPG and/or CDDP, were observed for the anti-tumor effect of the drugs and were subjected to immunohistochemical study. RESULTS: 1) Tumor reduction was enhanced by the combined use of SPG and CDDP; 2) The survival rate of rats given SPG alone was significantly higher than that of the control rats, and treatment with SPG combined with CDDP tended to improve the survival rate; 3) Immunohistochemically, an infiltrative increase in cytotoxic T-lymphocytes (CTL) was induced, although the development of helper T-cells and macrophages was immunohistochemically weak at the tumor site. CONCLUSION: The administration of SPG enhanced the anti-tumor effect of CDDP.     

Seasonal influence on 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis in Sprague-Dawley rats under controlled laboratory conditions

There is good evidence in some species, including rats, that circannual rhythms are innate and can occur even under constant environmental conditions. Such circannual rhythms, e.g. in hormone levels and immune system function, may influence tumourigenesis. This prompted us to study 7,12-dimethylbenz[a]anthracene(DMBA)-induced mammary carcinogenesis at different seasons of the year in female Sprague-Dawley rats under constant environmental conditions (photoperiod, temperature, air humidity, food). DMBA was administered orally at a dose of 5 mg per rat at the first day of the experiment and then at weekly intervals up to a total dose of 20 mg per rat. Rats were palpated once weekly for the presence of mammary tumours. After 13 weeks, they were necropsied for examination of the number and size of mammary tumours. Age-matched groups of 36-99 rats were used per experiment. When the experiment was performed twice within 2 years during the same season (spring/summer), tumour incidence (56 and 61%) and tumour burden were almost equal, indicating that data obtained in this way were reproducible. However, the same experiment performed in autumn yielded a significantly lower tumour incidence (34%) and tumour burden. When the experiment was started during winter, tumour incidence was similar to the spring/summer groups, but tumour burden was lower. The data indicate a seasonal variation in the development and growth of DMBA-induced breast cancer in Sprague-Dawley rats. One possible explanation for this phenomenon may be the seasonal variation in pineal melatonin production and immune function previously reported in rodents under constant environmental conditions.     

Effect of dietary fats and sesamin on the lipid metabolism and immune function of Sprague-Dawley rats

We examined the effect of three dietary fats, safflower oil (SAF) rich in linoleic acid, borage oil (BOR) rich in gamma-linolenic acid, and perilla oil (PER) rich in alpha-linolenic acid, on the lipid metabolism, and chemical mediator and immunoglobulin levels in Sprague-Dawley rats, as well as the dietary effect of sesame-derived antioxidative sesamin. The serum cholesterol, phospholipid, triglyceride, prostaglandin E2 level and splenic leukotriene B4 level were lower in the rats fed on BOR or PER than in those fed on SAF. SES feeding suppressed the expression of the lipid-decreasing effect of BOR, but not in the rats fed on PER. In respect of the fatty acid composition of the liver and spleen, PER feeding gave a lower arachidonic acid level, and higher eicosapentaenoic and docosahexaenoic acid levels than SAF feeding did, while the effect of BOR feeding was marginal. The effect of SES feeding on fatty acid composition was much smaller than that of dietary fats. In respect of immunoglobulin production, PER + SES feeding gave the lowest IgE productivity in the mesenteric lymph node lymphocytes. These results suggest that PER feeding regulated lipid metabolism and exerted an anti-allergic effect by a different mechanism from that with BOR feeding.     

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 frequency of the butyrylcholinesterase K mutation was calculated on the basis of data obtained by polymerase chain reaction primer-introduced restriction analysis (PCR-PIRA). The population sample was composed of 177 Brazilians: 95 whites of predominantly European ancestry and 82 admixed individuals (European and African origin). The frequencies--18.4 +/- 2.8% for whites and 17.1 +/- 2.9% for admixed--did not differ from those previously obtained in North America, Scotland, Japan, and Denmark. The occurrence of the K mutation in Europeans, East Asians, and Africans suggests a relatively old origin for this mutation, and the similar frequencies found in these populations may suggest the operation of selective forces.     

Induction of mammary tumors in virgin female BALB/c mice by single low doses of 7,12-dimethylbenz[a]anthracene

The induction of mammary tumors in virgin female inbred BALB/c mice after administration of 7,12-dimethylbenz[a]anthracene (DMBA) over a wide range of doses was studied. Mice were exposed at 12 weeks of age to single or multiple doses of DMBA ranging from 0.0025 to 12.0 mg by gastric intubation and were checked regularly for mammary tumors. The experiment was terminated when the mice were 800 days of age. In the dose range of 0.0025--0.125 mg DMBA, the incidence of mammary tumors was dose-dependent. At higher doses, the mammary tumor incidence became less dose-dependent and was nearly independent of doses above the 0.25-mg level. Analysis of the data for the rate of appearance of mammary tumors with age of the animals and for the age at death of non-mammary tumor-bearing animals indicated that in the low dose range induction of mammary tumors was the predominant effect of DMBA exposure, whereas at moderate to high doses the toxic and carcinogenic effects of DMBA on other tissues significantly influenced the final incidence of mammary tumors. Greater than 90% of the tumors that resulted from administration of low doses of DMBA were adenocarcinomas. In contrast, adenocarcinomas and adenoacanthomas were found in approximately equal proportions following administration of high doses of DMBA.     

A rice protein isolate alters 7,12-dimethylbenz[alpha]anthracene-induced mammary tumor development in female rats

The effects of a rice protein isolate (RPI) on 7,12-dimethylbenz[alpha]anthracene (DMBA)-induced mammary tumor progression were investigated in female Sprague-Dawley rats. At 6 weeks of age, rats were fed a casein, RPI or soybean protein isolate (SPI) diet, respectively. After 1 week, DMBA was administered orally at the dose of 30 mg/kg body weight. The mean tumor number per tumor-bearing rat at autopsy was significantly lower only in rats fed RPI than in those fed casein. Palpable tumors at the mid point of the experiment were significantly lower in rats fed RPI and SPI than in those fed casein. Serum estradiol-17 beta concentrations were lower in rats fed the SPI (but not in those fed RPI) than in those fed casein. In a further experiment, no differences were found in hepatic microsomal DMBA-arylhydrocarbon hydroxylase activity after 7 days of feeding the respective diets. These results suggest that RPI exerts its inhibitory effect on DMBA-induced mammary tumorigenesis irrespective of changes in circulating estrogens or modulation of hepatic DMBA metabolism.     

Black tea and mammary gland carcinogenesis by 7,12-dimethylbenz[a]anthracene in rats fed control or high fat diets

Epidemiological studies suggest that tea may reduce cancer risk, and in laboratory rodents, chemopreventive effects of tea or purified extracts of tea have been demonstrated in lung, gastrointestinal tract and skin. There is some evidence of chemoprevention by tea in the mammary gland, but the data are not conclusive. In order to evaluate more fully the possible influence of black tea on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary gland tumors in the female S-D (Sprague-Dawley) rat, three large studies were performed: experiment 1, tumorigenesis in rats fed AIN-76A diet and given 25 mg/kg DMBA and 1.25 or 2.5% whole tea extract or water to drink; experiment 2, tumorigenesis in rats given 15 mg/kg DMBA and the same diet and fluids as in experiment 1; experiment 3, tumorigenesis in rats fed control or HF (high fat, corn oil) diet and given 15 mg/kg DMBA and 2% tea or water to drink. Tea was given throughout the experiment; DMBA was given by gastric gavage at 8 weeks of age. There was no consistent effect of tea on tumorigenesis in rats fed AIN-76A diet; there was, however, evidence in experiment 3 of a reduction of tumorigenesis by tea in rats fed the HF diet. In experiment 3, rats fed the HF diet and given water showed the expected increase in tumor burden (number and weight) compared with rats fed control diet. However, rats fed the HF diet and given 2% tea showed no increase in tumor burden; their tumor burden was significantly lower than in rats fed the HF diet and given water (P < 0.01) and was not different from rats fed control diet and given water or tea. In addition, in experiment 3, the number of malignant tumors per tumor-bearing rat was increased by the HF diet in water-drinking rats (P < 0.01) but not in tea-drinking rats. Therefore, it appears that tea partially blocked the promotion of DMBA-induced mammary tumorigenesis by the HF diet.     

The influence of melatonin on metabolic changes in female rats induced by continuous irradiation and/or administration of 7,12-dimethylbenz/a/anthracene

OBJECTIVE: To investigate the influence of social deprivation upon the diet, dental health behaviours and dental health status of five-year-old children in Northern Ireland. DESIGN: Cross-sectional study. SETTING: Fifty-eight primary schools in North and West Belfast. SUBJECTS: A nine per cent (240) random sample of 2,666 five-year-old children resident and attending school in North and West Belfast. MAIN OUTCOME MEASURES: Dental health status measured by dmft index; parental assessments of the child's dental health behaviours: parental dental attendance patterns and attitudes towards dental health; parental assessments of the child's diet and snacking behaviours: parental attitudes towards snack foods; and demographic profile of child's family. RESULTS: One hundred and sixty-three children were examined, a 68 per cent response. The majority of the families were either in low income employment or unemployed. Sixty-eight per cent of children had experience of dental caries. Dental decay was unrelated to parental employment status but more children from unemployed families attended when in pain. The diet of the children was related to both employment status and parental attitude and was reflected in their caries experience. Caries experience was dependent upon parental dental attendance, the consumption of carbonated drinks and sugar containing bedtime drinks. CONCLUSIONS: The results suggest that unemployment and parental attitudes are important as determinants of dental caries in five-year-old children from North and West Belfast. These factors may influence the child's dental health care as well as type of diet.     

Demonstration and partial characterization of a 7,12-dimethylbenz[alpha]anthracene-binding protein in rat adrenal

Rat adrenal cytosol was found to contain a 7,12-dimethylbenz[a]-anthracene (DMBA)-binding protein which is characterized by a pI of 7.2, a Kd-value of 3 microM and a maximal capacity of about 47 pmol/mg protein. The binding is highly specific for DMBA and is not displaced by 3-methylcholanthrene (MC), benz[a]pyrene (BP) or other polycyclic hydrocarbons. Likewise, various androgens, estrogens or glucocorticoids have no effect on the DMBA binding. It is proposed that the DMBA-binding protein may have a role in the toxic effects of DMBA or DMBA metabolites in adrenal and possibly in other DMBA-sensitive organs as well.     

Effects of 7,12-dimethylbenz[a]anthracene on the superantigen toxic shock syndrome toxin (TSST-1)-induced proliferation and antibody secretion by human lymphocytes

Toxic shock syndrome toxin (TSST-1), a 22-kDa exotoxin secreted by Staphylococcus aureus, can act as a nominal antigen and induce proliferation and immunoglobulin secretion in human B-cells. The purpose of the present studies was to examine the effect of 7,12-dimethylbenz[a]anthracene (DMBA), a well-characterized immunosuppressant of both cell-mediated and humoral immunity in murine lymphocytes, upon the mixed lymphocyte reaction (MLR) and TSST-1-induced immune responses in human lymphocytes. The MLR, using human tonsillar lymphocytes (HTL) from four different donors, was inhibited in a dose-dependent manner from 1 to 100 microM. The IC50 for the suppression of the MLR ranged from 10 to 40 microM. TSST-1 is a potent stimulator of T-cells bearing specific VB regions on the T-cell receptor (TCR). In contrast with the results from the MLR, DMBA inhibited TSST-1-induced T-cell proliferation only at 100 microM in HTL. A similar profile of activity was determined with splenic T-cells from a single donor. TSST-1 has also been demonstrated to induce specific B-cell proliferation and differentiation in the presence of irradiated T-cells. TSST-1-induced B-cell proliferation was only consistently and markedly inhibited by DMBA at 100 microM in tonsillar and splenic lymphocytes. In contrast, TSST-1-induced B-cell differentiation, as manifested by IgM and IgG secretion, was inhibited in a dose-dependent manner from 1 to 100 microM DMBA in B-cells from human tonsils and spleens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antitumour activity of coumarin and 7-hydroxycoumarin against 7,12-dimethylbenz[a]anthracene-induced rat mammary carcinomas

Female SD rats with established 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumours were treated with coumarin (20 mg/kg body weight; six times per week) or its metabolite 7-hydroxycoumarin (20 mg/kg) for 4 weeks. The anti-oestrogen tamoxifen (8.8 mg/kg) served as the reference drug. The inhibitory effect of coumarin was similar to that of tamoxifen [mean change of tumour area: 428% (coumarin) compared to 528% (tamoxifen); control 822%]. The strongest inhibition was observed with 7-hydroxycoumarin (248%); the difference compared to the control was significant (P < 0.01). Neither coumarin nor 7-hydroxycoumarin reduced the number of tumours appearing during treatment as tamoxifen did. However, the size of the tumours treated with coumarin or its metabolite was generally much smaller than those in the tamoxifen group or in the control group. From the data obtained it appears that coumarin and 7-hydroxycoumarin inhibit the growth of tumours that have reached a certain size but do not prevent the formation of tumours after exposure to the carcinogen.     

Metabolism of a carcinogen (7,12-dimethylbenzanthracene) in the pancreas of the Long-Evans rat

Because of the high incidence of pancreatic cancer in the United States and because of the correlation of pancreatic cancer to environmental exposure, we have undertaken experiments to measure the metabolism of the carcinogen 7,12-dimethylbenzanthracene in the pancreas of the Long-Evans male rat. This study examined the in vitro metabolism of the carcinogen and found the production of aqueous products in pancreas to be similar to that in liver, however, the pancreatic capability was not induced to greater metabolism by pretreatment with either phenobarbital or 3-methylcholanthrene. High pressure liquid chromatographic analysis of the in vitro products of pancreatic metabolism demonstrated a relatively greater abundance of 5,6-epoxy-7-hydroxymethyl-12-methylbenzanthracene than the liver and a relatively less abundance of 7-hydroxymethyl-12-methylbenzanthracene and 7-methyl-12-hydroxymethylbenzanthracene than the liver. Carcinogen levels were measured in pancreas, liver, bile and blood at 2, 5, 10, 16, 22 and 36 hours after injection.