Induction of cell proliferation, micronuclei and hyperdiploidy/polyploidy in the mammary cells of DDT- and DMBA-treated pubertal rats

The environmental estrogen, dichlorodiphenyltrichloroethane (DDT), and its metabolites have been implicated in the development of breast cancer through mechanisms that remain to be elucidated. It has been hypothesized that exposure to DDT and its metabolites, during critical periods of development, can contribute to an elevated risk for breast cancer in adults. In the present study, we have investigated the effect of o,p'-DDT on mammary gland cell proliferation and chromosomal alterations, in a rat mammary cancer model (commonly used to study human cancer), to gain insights into its potential role in the development of breast cancer. Twenty-one-day-old female Sprague-Dawley (SD) rats were administered o,p'-DDT, 7,12-dimethylbenz[a]anthracene (DMBA), genistein, DDT+DMBA, or DDT+DMBA+genistein, over a 14-day period. To determine changes in chromosome number and structure, we used the micronucleus assay as well as multicolor fluorescence in situ hybridization (FISH) region-specific DNA probes for rat chromosomes 4 and 19. Cell proliferation was evaluated using 5-bromo-2'-deoxyuridine (BrdU). Significant increases in BrdU-incorporated cells were seen in the rats treated with DDT+DMBA. Although micronucleus frequencies were somewhat elevated in several of the treatment groups, significant increases were not seen in any of them. Significant increases in numerical chromosomal aberrations were detected in all of the DDT- and DMBA-treated groups. Genistein significantly reduced BrdU incorporation and polyploidy in the DDT+DMBA-treated rats. These initial studies indicate that DDT and DMBA can induce cellular and chromosomal alterations in the rat mammary gland, which is consistent with the hypothesis that these agents can induce early events in mammary carcinogenesis.     

Protective Role of Withaferin-A on Red Blood Cell Integrity During 7,12-Dimethylbenz[a]anthracene Induced Oral Carcinogenesis

The aim of the present study was to investigate the protective effect of Withaferin-A on red blood cell integrity during 7,12-dimethylbenz[a]anthracene (DMBA) induced oral carcinogenesis. The protective effect of Withaferin-A was assessed by measuring the status of glycoconjugates, membrane bound enzyme activity and red blood cell osmotic fragility. Oral squamous cell carcinoma was induced in the buccal pouch of Syrian golden hamsters by painting with 0.5% DMBA in liquid paraffin thrice a week for 14 weeks. The levels of glycoconjugates, membrane bound enzyme activity, osmotic fragility and thiobarbituric acid reactive substances (TBARS) were analyzed by using specific colorimetric methods. We observed 100% tumor formation in DMBA painted hamsters. Increase in plasma glycoconjugates at the expense of red blood cell membrane glycoconjugates levels were observed in DMBA painted hamsters as compared to control hamsters. Erythrocytes from DMBA painted hamsters were more fragile than those from control hamsters. The activity of membrane bound enzyme (Na⁺ K⁺ ATPase) decreased whereas TBARS level was increased in DMBA painted hamsters as compared to control hamsters. Oral administration of Withaferin-A at a dose of 20mg kg⁻¹ bw significantly prevented the tumor formation as well as normalized the biochemical variables in DMBA painted hamsters. Our results thus demonstrate the protective effect of Withaferin-A on red blood cell integrity during DMBA induced oral carcinogenesis.     

Clerodendron Inerme Protects Cellular Integrity during 7,12-Dimethylbenz[A]-Anthracene Induced Hamster Buccal Pouch Carcinogenesis

Aim of the present study was to investigate the protective effect of Clerodendron inerme on cellular integrity by measuring the status of glycoconjugates, lipids, osmotic fragility, and membrane bound enzyme activity in 7, 12-dimethylbenz (a) anthracene (DMBA)-induced oral carcinogenesis. Oral squamous cell carcinoma was induced in the buccal pouch of Syrian golden hamsters by painting with 0.5% DMBA in liquid paraffin thrice a week for 14 weeks. The levels of glycoconjugates, lipids, osmotic fragility and membrane bound enzyme activity were analyzed by using specific colorimetric methods. We observed 100% tumor formation in DMBA painted hamsters. Altered glycoconjugates and lipid pattern were observed in DMBA painted hamsters as compared to control hamsters. Erythrocytes from DMBA painted hamsters were more fragile than those from control hamsters. The activity of membrane bound enzyme (Na⁺ K⁺ ATPase) decreased in DMBA painted hamsters as compared to control hamsters. Oral administration of aqueous leaf extract of Clerodendron inerme (CiALet) at a dose of 500mg/kg body weight significantly prevented the tumor formation and histopathological abnormalities as well as normalized the above said biochemical variables in DMBA painted hamsters. Our results thus demonstrate the protective effect of Clerodendron inerme on cellular integrity during DMBA induced oral carcinogenesis.     

Endogenous estrogen status, but not genistein supplementation, modulates 7,12-dimethylbenz[a]anthracene-induced mutation in the liver cII gene of transgenic big blue rats

A growing number of studies suggest that isoflavones found in soybeans have estrogenic activity and may safely alleviate the symptoms of menopause. One of these isoflavones, genistein, is commonly used by postmenopausal women as an alternative to hormone replacement therapy. Although sex hormones have been implicated as an important risk factor for the development of hepatocellular carcinoma, there are limited data on the potential effects of the estrogens, including phytoestrogens, on chemical mutagenesis in liver. Because of the association between mutation induction and the carcinogenesis process, we investigated whether endogenous estrogen and supplemental genistein affect 7,12-dimethylbenz[a]anthracene (DMBA)-induced mutagenesis in rat liver. Intact and ovariectomized female Big Blue rats were treated with 80 mg DMBA/kg body weight. Some of the rats also received a supplement of 1,000 ppm genistein. Sixteen weeks after the carcinogen treatment, the rats were sacrificed, their livers were removed, and mutant frequencies (MFs) and types of mutations were determined in the liver cII gene. DMBA significantly increased the MFs in liver for both the intact and ovariectomized rats. While there was no significant difference in MF between the ovariectomized and intact control animals, the mutation induction by DMBA in the ovariectomized groups was significantly higher than that in the intact groups. Dietary genistein did not alter these responses. Molecular analysis of the mutants showed that DMBA induced chemical-specific types of mutations in the liver cII gene. These results suggest that endogenous ovarian hormones have an inhibitory effect on liver mutagenesis by DMBA, whereas dietary genistein does not modulate spontaneous or DMBA-induced mutagenesis in either intact or ovariectomized rats.     

Influence of Strain and Sex on the Local Development of Mammary Tumors Induced by Direct Application of DMBA Powder to Rat Mammary Glands

In order to determine the influence of strain and sex on local carcinogenesis in rat mammary tissue, 1 mg of 7, 12 dimethylbenz(a)anthracene (DMBA) was dusted directly onto the exposed mammary gland of 30-day-old Long-Evans (L-E) rats and Sprague-Dawley (S-D) rats. The experiment was terminated 28 weeks after application of the carcinogen. Tumors measuring between 1 and 2 cm in diameter were harvested from female L-E rats with high frequency (85%) and long latency (mean: 23.7 weeks after DMBA dusting), and from female S-D rats with extremely high frequency (98%) and short latency (16.7 weeks). Male rats of both strains were almost identically much less susceptible to DMBA (L-E; 55%, 25.0 weeks, S-D; 53%, 23.9 weeks). Ovariectomized S-D (47%, 24.9 weeks) and orchiectomized S-D (30%, 24.8 weeks) rats, which were gonadectomized at 30 days of age, respectively, were also much less susceptible. A variety of histologies, mostly malignant epithelial, mesenchymal or mixed tumors, were noted in each group. The carcinomatous response in the mammary tissue was much higher in female S-D (96%) than in female L-E (50%) rats, and very low in male and gonadectomized rats (10-20%). In contrast, the sar-comatous response in the mammary tissue was moderate in female and male L-E and male S-D (43-50%) rats, and low in the other groups (15-29%). Acta Pathol Jpn 40: 9–13, 1990.     

Influence of strain and sex on the local development of mammary tumors induced by direct application of DMBA powder to rat mammary glands

In order to determine the influence of strain and sex on local carcinogenesis in rat mammary tissue, 1 mg of 7,12-dimethylbenz(a)anthracene (DMBA) was dusted directly onto the exposed mammary gland of 30-day-old Long-Evans (L-E) rats and Sprague-Dawley (S-D) rats. The experiment was terminated 28 weeks after application of the carcinogen. Tumors measuring between 1 and 2 cm in diameter were harvested from female L-E rats with high frequency (85%) and long latency (mean: 23.7 weeks after DMBA dusting), and from female S-D rats with extremely high frequency (98%) and short latency (16.7 weeks). Male rats of both strains were almost identically much less susceptible to DMBA (L-E; 55%, 25.0 weeks, S-D; 53%, 23.9 weeks). Ovariectomized S-D (47%, 24.9 weeks) and orchiectomized S-D (30%, 24.8 weeks) rats, which were gonadectomized at 30 days of age, respectively, were also much less susceptible. A variety of histologies, mostly malignant epithelial, mesenchymal or mixed tumors, were noted in each group. The carcinomatous response in the mammary tissue was much higher in female S-D (96%) than in female L-E (50%) rats, and very low in male and gonadectomized rats (10-20%). In contrast, the sarcomatous response in the mammary tissue was moderate in female and male L-E and male S-D (43-50%) rats, and low in the other groups (15-29%).     

The effects of neonatal androgenization on mammary gland mitotic rate and susceptibility of carcinogen-induced mammary dysplastigenesis and tumorigenesis in LEW/Mai rats.

The influence of neonatal testosterone propionate treatment (androgenization) on mammary gland mitotic rate (MR) and susceptibility to 7,12-dimethylbenz(alpha)anthracene (DMBA) carcinogenesis was studied in female LEW/Mai rats. Mammary gland MR in androgenized rats was significantly lower than MR in normal rats at all ages studied. Treatment of androgenized rats with DMBA resulted in a significant increase in mammary gland MR in comparison with untreated androgenized rats. MR in DMBA-treated androgenized rats was similar to MR in DMBA-treated normal rats at most intervals after the introduction of the carcinogen. Although mammary epithelial MR in androgenized rats was significantly lower than that of normal rats of comparable age, no evidence of a decrease in susceptibility of mammary epithelium in androgenized rats to DMBA carcinogenesis was found. Instead, androgenized rats had a higher incidence of DMBA-induced mammary dysplasias, with no change in their morphologic or histologic features, than did normal rats; and there was no change in the incidence, latency, or histopathologic appearance of DMBA-induced mammary tumors in androgenized versus normal rats.     

Restorative effect of Dendrophthoe falcata (L.f.) Ettingsh on lipids,lipoproteins, and lipid-metabolizing enzymes in DMBA-induced mammary gland carcinogenesis in Wistar female rats

Lipid, lipoproteins, and lipid-metabolizing enzymes are associated with breast cancer risk. In this study, the potential of hydroalcoholic extract (HEDF) of Dendrophthoe falcata (L.f) Ettingsh (Loranthaceae) for the management of lipid metabolism on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma was investigated in Wistar female rats. Thirty female rats (55 days of age) were divided into five groups: control, DMBA (25 mg in 0.5 ml of olive oil by air pouch technique), DMBA?+?HEDF (250, 475, and 950 mg/kg). After 90 days of induction, HEDF was administered for 28 days by gastric intubations. The levels of lipids, lipid-metabolizing enzymes, and lipoproteins were analyzed in the plasma and liver of both control and experimental animals. Rats treated with DMBA showed an increase (p?<?0.05) in levels of phospholipids, triglycerides, free fatty acids, total cholesterol, and free cholesterol and a decrease (p?<?0.01) in levels of ester cholesterol in the plasma and liver. The levels of very low-density lipoprotein and low-density lipoprotein increased (p?<?0.01), while the levels of high-density lipoproteins decreased (p?<?0.001) in plasma. Moreover, there was a significant increase (p?<?0.001) in activities of total lipase, cholesterol ester hydrolase, cholesterol ester synthase but with a sharp decrease (p?<?0.01) in lecithin cholesterol-acyl transferase and lipoprotein lipase in animals with mammary cancer. HEDF treatment caused the activity of these alterations in biochemical parameters to return to almost normal control levels. Also, histopathological analysis of the breast tissue was analyzed by hematoxylin and eosin staining, and results revealed the cytoprotective role of HEDF against mammary carcinogenesis. The effects of HEDF were found to be dose dependent in nature.     

Effects of progesterone on mammary carcinogenesis when various doses of DMBA were applied directly to rat mammae

To investigate the suggestion that progesterone acts directly on the mammary gland to inhibit carcinogenesis, doses of 500, 100 or 30 microgram of DMBA were applied directly to inguinal mammary glands of 50 d old rats which either received no other treatment or were injected with progesterone (3 mg/d) s.c. for 18 d before dusting the carcinogen. Progesterone pretreatment did not inhibit mammary carcinogenesis in rats dusted with 500 microgram or DMBA. When smaller doses of DMBA (100 microgram or less) were applied, hormone pretreatment markedly reduced carcinogenesis, the inhibitory effect being statistically significant in the group dusted with the smallest dose of carcinogen. As the dusting technique eliminated any observable systemic effects of the carcinogen, it is concluded that the results support the suggestion that progesterone acts directly on the mammary gland to inhibit carcinogenesis and that this effect can be over-ridden if a large enough dose of DMBA (somewhere between 100 and 500 micrograms) is applied. The importance of carcinogen dose to resulting tumour yield was clearly shown by the significant descending gradation in tumour incidences and gradual lengthening of average tumour latent periods in the 3 control groups with decreasing DMBA dose, as well as in the 3 hormone-pretreated groups.     

A proposed selective cell carcinogenesis in mammary tumors.

The present study was done to ascertain whether a specific carcinogenic agent has a causal effect on the initial proliferation of only one cell type or whether it acts indiscriminately on all cells in the breast secretory unit. Enzymes histochemistry and electron microscopy were performed on DMBA-induced mammary tumors in female Sprague-Dawley rats and on virus-associated spontaneous mammary tumors in C3H/HEJ mice. The results showed that the chemical carcinogen DMBA affects initial myoepithelial cell proliferation, while virus-associated mammary carcinoma originated from ductular epithelial cell proliferation. To determine whether a specific tumor is composed of a single cell type, tumors were grown in tissue culture. The monolayer was fixed in the usual manner for electron microscopy while in Falcon tissue culture plates. The plates were dissolved in xylene and the monolayer was cut into small pieces and embedded in the plastic media. Electron microscopy performed on the tissue culture and the original tissue from the virus-induced tumors showed the presence of viruses in large numbers. It also suggested the differentiation of basal membrane to form basal lamina and apical plasma membrane into microvilli. This study strongly suggests the presence of selective cell carcinogenesis in the mammary gland.     

Evaluation of carcinogenic potential of diuron in a rat mammary two-stage carcinogenesis model

This study aimed to evaluate the carcinogenic potential of the herbicide Diuron in a two-stage rat medium-term mammary carcinogenesis model initiated by 7,12-dimethylbenz(a)anthracene (DMBA). Female seven-week-old Sprague-Dawley (SD) rats were allocated to six groups: groups G1 to G4 received intragastrically (i.g.) a single 50 mg/kg dose of DMBA; groups G5 and G6 received single administration of canola oil (vehicle of DMBA). Groups G1 and G5 received a basal diet, and groups G2, G3, G4, and G6 were fed the basal diet with the addition of Diuron at 250, 1250, 2500, and 2500 ppm, respectively. After twenty-five weeks, the animals were euthanized and mammary tumors were histologically confirmed and quantified. Tumor samples were also processed for immunohistochemical evaluation of the expressions of proliferating cell nuclear antigen (PCNA), cleaved caspase-3, estrogen receptor-α (ER-α), p63, bcl-2, and bak. Diuron treatment did not increase the incidence or multiplicity of mammary tumors (groups G2 to G4 versus Group G1). Also, exposure to Diuron did not alter tumor growth (cell proliferation and apoptosis indexes) or immunoreactivity to ER-α, p63 (myoephitelial marker), or bcl-2 and bak (apoptosis regulatory proteins). These findings indicate that Diuron does not have a promoting potential on mammary carcinogenesis in female SD rats initiated with DMBA.     

Therapeutic potential of Dendrophthoe falcata (L.f) Ettingsh on 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis in female rats: effect on antioxidant system, lipid peroxidation, and hepatic marker enzymes

In this study, the therapeutic potential of the hydroalcoholic extract of Dendrophthoe falcata (L.f) Ettingsh (Loranthaceae; HEDF) on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma was investigated in Wistar female rats at 55 days of age. Thirty female rats were divided into five groups with six animals in each group: control, DMBA (25 mg in 0.5 ml olive oil by air pouch technique), and DMBA + HEDF (250, 475, and 950 mg/kg). After 90 days of induction, HEDF were administered for 28 days, by gastric intubations. The levels of lipid peroxides and activities of enzymic and nonenzymic antioxidants were measured in serum, liver, kidney, and breast of both control and experimental groups. In addition to this, liver marker enzymes were also assessed. Rats treated with DMBA showed an increase in lipid peroxidation accompanied by high malondialdehyde levels along with lowered activities of enzymic antioxidants (superoxide dismutase, catalase, glutathione peroxidase) and nonenzymic antioxidants (glutathione, ascorbic acid, and α-tocopherol). A significant decrease in alanine aminotransferase, aspartate aminotransferase with a sharp increase in alkaline phosphatase, acid phosphatase, and 5′-nucleotidase was observed in the liver of mammary cancer-bearing animals. HEDF treatment caused the activity of these liver marker enzymes’ return to almost normal control levels. Furthermore, the breast tumor weight decreased significantly in the DMBA + HEDF-treated groups. This result suggests that HEDF shows antioxidant activity and play a protective role against DMBA-induced breast carcinogenesis.     

DNA damage induced by 7,12-dimethylbenz[a]anthracene in the liver and the mammary gland of rats exposed to polycyclic aromatic hydrocarbon enzyme inducers during perinatal life

The long-lasting modulating effect induced by the prenatal or neonatal exposure to phenobarbital (PB) and aroclor on the genotoxic activity of 7,12-dimethylbenz[a]anthracene (DMBA) in female Sprague-Dawley rats was studied. The effect was measured as DNA damage evaluated in the liver and in the mammary gland of 55-day-old animals, 4 and 24 h after an i.g. injection of 80 mg/kg of DMBA. PB was given per os, i.g. or in drinking water to pregnant females and by i.g. only to neonates or in adult progeny. Aroclor was injected i.g. in prenatal and in neonatal life, and a second dose was given in adult life. Under these experimental conditions it was shown that DNA damage kinetics caused by DMBA are modulated by exposure to PB and, to a minor extent, by aroclor. The amount and persistence of DNA damage were highest when PB was administered to neonates. An average 2-fold increase in the elution constants (K) of DNA in the liver and the mammary gland was observed 4 h after DMBA treatment, as compared to uninduced animals. Repeated enzyme induction by PB seems to reduce DMBA genotoxicity, as shown by a decrease in DNA damage and persistence in the liver and mammary gland. The inducibility of the monooxygenase enzyme system in perinatal life favouring metabolic activation or inactivation of polycyclic aromatic hydrocarbons might be critical in determining individual susceptibility of adult progeny to chemical carcinogenesis by DMBA.     

Individual and combined soy isoflavones exert differential effects on metastatic cancer progression

To investigate the effects soy isoflavones in established cancers, the role of genistein, daidzein, and combined soy isoflavones was studied on progression of subcutaneous tumors in nude mice created from green fluorescent protein (GFP) tagged-MDA-MB-435 cells. Following tumor establishment, mice were gavaged with vehicle or genistein or daidzein at 10 mg/kg body weight (BW) or a combination of genistein (10 mg/kg BW), daidzein (9 mg/kg BW), and glycitein (1 mg/kg BW) three times per week. Tumor progression was quantified by whole body fluorescence image analysis followed by microscopic image analysis of excised organs for metastases. Results show that daidzein increased while genistein decreased mammary tumor growth by 38 and 33% respectively, compared to vehicle. Daidzein increased lung and heart metastases while genistein decreased bone and liver metastases. Combined soy isoflavones did not affect primary tumor growth but increased metastasis to all organs tested, which include lung, liver, heart, kidney, and bones. Phosphoinositide-3-kinase (PI3-K) pathway real time PCR array analysis and western blotting of excised tumors demonstrate that genistein significantly downregulated 10/84 genes, including the Rho GTPases RHOA, RAC1, and CDC42 and their effector PAK1. Daidzein significantly upregulated 9/84 genes that regulate proliferation and protein synthesis including EIF4G1, eIF4E, and survivin protein levels. Combined soy treatment significantly increased gene and protein levels of EIF4E and decreased TIRAP gene expression. Differential regulation of Rho GTPases, initiation factors, and survivin may account for the disparate responses of breast cancers to genistein and daidzein diets. This study indicates that consumption of soy foods may increase metastasis.     

Mutant frequency and molecular analysis of in vivo lacI mutations in the bone marrow of Big Blue rats treated with 7, 12-dimethylbenz[a]anthracene

Recently, we evaluated lacI mutations in lymphocytes and mammary tissue of Big Blue (BB) rats exposed to 7, 12-dimethylbenz[a]anthracene (DMBA). The results on the time course of mutant induction suggested that the lacI gene may manifest a tissue-specific increase in mutant frequency (MF). To test whether a tissue-specific increase in lacI MF is dependent on the cell proliferation rate of a tissue, we examined rapidly proliferating bone marrow cells for DMBA-induced lacI mutations. Seven-week-old female BB rats were given single doses of 0, 20, and 130 mg/kg DMBA by gavage and the lacI MFs in the bone marrow were measured over a period of 14 weeks following treatment. Bone marrow cells had a remarkably low average background MF (3.1 +/- 1.6 x 10(-6) plaque-forming units) and the DMBA-induced lacI MFs were significantly higher than control MFs for both doses and at all time points (P < 0.01). The lacI MF in the bone marrow increased for 2 weeks and then remained relatively constant; 20 and 130 mg/kg DMBA produced 34- and 106-fold increases in MF over control MF. DNA sequencing revealed that the majority of DMBA-induced lacI mutations were base-pair substitutions and that A:T --> T:A (48%) and G:C --> T:A (24%) transversions were the predominant types. Thus, the different lacI mutation fixation times observed for bone marrow (2 weeks), mammary (10 weeks), and lymphocytes (6 weeks) suggest that the lacI gene manifests a tissue-specific mutation fixation time, which may depend on the cell proliferation rate of a tissue. In addition, the relatively low spontaneous MF in bone marrow compared with that in other tissues may be useful for increasing the sensitivity of the assay for detecting induced MFs in BB rats.     

Ameliorating effect of phytoestrogens on CCl4-induced oxidative stress in the livers of male Wistar rats

Glutathione-S-transferases and glutathione play a key role in the detoxification of most toxic agents. In the present study, the protective effects, if any, of isoflavone phytoestrogens--genistein and daidzein on the carbon tetrachloride (CCl4) induced changes in the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutathione S transferase (GSH) and levels of glutathione (GSH) and thiobarbituric acid reactive substances (TBARS)-were studied. The activities of ALT and AST were assayed in the serum, whereas the activity of GST and levels of GSH and TBARS were determined in the livers of rats. The current study involved the division of animals into two main groups: (i) rats pretreated with genistein and daidzein for three days; and (ii) non-pretreated rats. In the pretreated group, rats received oral doses of genistein (7.9 micromol/kg body weight) and daidzein (7.9 micromol/kg body weight) for three consecutive days (once daily) followed by oral dose of CCl4 on the 4th and the 5th day concurrently with the phytoestrogens-genistein or daidzein. In the non-pretreated group animals received oral dose of CCl4 (1 ml/kg body weight) for two consecutive days along with the phytoestrogens-genistein or daidzein. Treatment of male rats with CCl4 significantly elevated the activity of ALT and AST in serum and levels of TBARS in the liver. On the other hand, CCl4 resulted in decreased activity of GST and lowered the GSH levels. Coadministration of genistein and daidzein with CCl4 could not restore the alterations in the activity of ALT and AST caused by CCl4 to normal control levels. However, repeated dose treatments with genistein and daidzein for three days prior to the administration of CCl4 restored such alterations to normal levels. Our results indicate that genistein is more effective than daidzein in counteracting the inhibition of GST activity caused by CCl4 and restoring it to normal levels. Genistein was also more effective than daidzein restoring the induced TBARS levels caused by CCl4 to normal control levels when rats were pretreated with the isoflavone orally for three days. It has been observed that the tested isoflavonoids were able to antagonize the toxic effects of CCl4. Such counteracting effects were more pronounced for genistein and when the phytoestrogens were administered as repeated doses prior CCl4 administration.     

Influence of surface lipids on skin carcinogenesis in rats.

Skin tumours were indeuced in female Wistar SPF-rats by different polycyclic aromatic hydrocarbons, benzo[alpha]pyrene (BP), dibenz[alpha, h]anthracene (DBA), 7,12-dimethylbenz]alpha]anthracene (DMBA), and 3-methylcholanthrene (MCA) being applied on the dorsal skin of 20 rats each. DBA was not carcinogenic under the experimental conditions. DMBA proved the most potent carcinogen, and MCA was more potent than BP. Half of the animals in each group underwent skin-surface lipid extraction (SSLE) before the application of carcinogen. SSLE did not influence the cumulative number of rats with skin tumours during an observation period of 15 months nor the type of tumours induced. The lipid extraction, however, increased the latency period and decreased the rate of tumour development when BP and MCA acted as carcinogens. On the contrary, SSLE enhanced the rate of tumour production by DMBA and reduced the latency period. The role of sebum and its composition in skin carcinogenesis is discussed, and an explanation of the different influence of SSLE on BP and MCA carcinogenesis is discussed, and and explanation of the different influence of SSLE on BP and MCA carcinogenesis in contrast to DMBA carcinogenesis is sought in differences in metabolic activation of the carcinogens.     

STUDIES ON TUMOR INDUCTION IN RATS WITH 7,12–DIMETHYL-BENZ (a) ANTHRACENE, WITH SPECIAL REFERENCES TO THE MORPHOGENESIS AND FINE-STRUCTURAL CHANGES IN THE TARGET ORGANS

After multiple injections of 7,12–DMBA (25 mg/kg body weight), high yields of malignant tumors were obtained in two different strains of rats (Donryu and ACI). The incidence of mammary cancer was high in female rats of both strains (84.6% in Donryu, 89.5% in AC I). High yields ( 67.5% ) of ear duct cancer were obtained in ACI males, and the incidence of leukemia was highest in Donryu females (46.296).
Changes produced with 7, 12–DMBA in several organs were observed by electron microscopy. Acute changes in the organs in which a malignant tumor could be induced appeared irreversible, including steady increase of tonofilaments in the ear duct glandular cell and appearance of numerous plasma cells in the spleen. The changes produced in the liver and kidney were found to be reversible.
Morphological discontinuity at the malignant transformation was recognized in the ear duct gland and spleen.
Ultrasturctures of induced malignant tumor cells were studied, and mammary cancer was classified into a highly differentiated type and a poorly differentiated type.
Classification of organs into three categories, a) DMBA-responding organ, b) DMBA-metabolizing and DMBA-susceptible organ, c) DMBA-refractory organ, was proposed and each was discussed in detail. ACTA PATH. JAP. 18 : 51–72, 1968.     

Morphological and biological characteristics of mammary tumours induced by the direct application of DMBA powder to rat mammary glands

Summary Mammary tumours were induced by the direct dusting of 1 mg, 7,12-dimethylbenz(a)anthracene (DMBA) powder onto the mammary gland of both 30-day-old female and male Sprague-Dawley rats, and the tumours were examined histologically. Mammary tumours developed in 43/43 (100%) of the females 11 to 20 weeks after DMBA dusting and 16/23 (70%) of the males 18 to 28 weeks after dusting, while non-mammary spindle cell sarcomas occurred in 5/23 (22%) of the males 15 to 24 weeks after dusting. A variety of benign and malignant mammary tumours of epithelial and/or mesenchymal origin were induced, which are comparable to human mammary tumours. Different histological patterns were observed in different areas of the same tumours. Ovariectomy revealed hormone (ovary)-dependency in 10/17 (59%) of the tumours, revealing regressing epithelial and proliferating mesenchymal tumour elements on histological examination.This work is supported in part by grant-in-aid for cancer research 63010020, from the Ministry of Education, Science, and Culture, Japan     

(1)H nuclear magnetic resonance metabolomic analysis of mammary tumors from lean and obese Zucker rats exposed to 7,12-dimethylbenz[a]anthracene

Obesity increases mammary tumor development in Zucker rats following a single administration of the procarcinogen 7,12-dimenthylbenz(a)anthracene (DMBA). Fifty-day-old obese and lean female Zucker rats were orally gavaged with 65 mg/kg DMBA and sacrificed 139 days post DMBA treatment. At the end of the experiment, mammary tumors were detected in 68% of the obese rats compared to 32% of the lean group (P<0.001). 1H nuclear magnetic resonance (1H-NMR) spectra obtained for hydrophilic and lipophilic extracts from excised tumors illustrated fundamental differences in metabolic profiles between the two groups. Differences were observed for key choline compounds, namely phosphocholine and glycerophosphocholine, both markers of malignancy and apoptosis. In addition, levels of lactate, creatine, myo-inositol, alpha-glucose, alanine, leucine, glutamate, glutamine, tyrosine, phenylalanine, and NADH varied between the lean and obese groups. Principal component analysis indicated class separation between tumors from lean and obese rats based on their metabolic profiles, illustrating the potential for using 1H-NMR metabolomic methods for identifying altered metabolic pathways. Our results suggest that obesity enhances the risk for DMBA-induced mammary tumor development in rats. However, the mechanism for this increase in risk is currently unknown and will require further studies for elucidation.