Development Of 7,12-dimethylbenz[a]anthracene Research Articles

Oral administration of tartrazine (E102) accelerates the incidence and the development of 7,12-dimethylbenz(a) anthracene (DMBA)-induced breast cancer in rats

BackgroundDespite the considerable advances made in the treatment of cancer, it remains a global threat. Tartrazine (E102) is a synthetic dye widely used in food industries; it has recently been shown to induce oxidative stress (a well known risk factor of cancer) in rat tissues. The present work therefore aimed to assess the impact of a regular consumption of tartrazine on the incidence of breast cancer in rats.MethodsForty (40) Wistar rats aged 55 to 60 days were randomly assigned into 5 groups (n = 8) including two groups serving as normal controls and receiving distilled water (NOR) or tartrazine (NOR + TARZ). The three remaining groups were exposed to the carcinogen DMBA (50 mg/kg) and treated for 20 weeks with either distilled water (DMBA), tartrazine 50 mg/kg (DMBA + TARZ) or a natural dye (DMBA + COL). The parameters evaluated were the incidence, morphology and some biomarkers (CA 15–3, estradiol and α-fetoprotein) of breast cancer. The oxidative status and histomorphology of the tumors were also assessed.ResultsA regular intake of tartrazine led to an early incidence of tumors (100% in rats that received TARZ only vs 80% in rats that received DMBA only), with significantly larger tumors (p < 0.001) (mass = 3500 mg/kg and volume = 4 cm3). The invasive breast carcinoma observed on the histological sections of the animals of the DMBA + TARZ group was more developed than those of the DMBA group. The increase in serum α-fetoprotein (p < 0.05) and CA 15–3 (p < 0.01) levels corroborate the changes observed in tumors. The presence of oxidative activity in animals of the DMBA + TARZ group was confirmed by a significant decrease (p < 0.001) in the activity of antioxidant enzymes (SOD and catalase) as well as the level of GSH and increase in the level of MDA compared to the rats of the DMBA and NOR groups.ConclusionTartrazine therefore appears to be a promoter of DMBA-induced breast tumorigenesis in rats through its oxidative potential. This work encourages further studies on the mechanisms of action of tartrazine (E102) and its limits of use.

Isoflavone and its metabolite equol inhibit the development of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumours in ovariectomised rats

Studies have yet to determine whether or not equol can explain the chemopreventive effect of soy. In the present study, a single oral dose of 5mg 7,12-dimethylbenz(a)anthracene (DMBA) was administered to 120 rats. After ovariectomy, these rats were divided into eight different treatment groups: control group (CG); high isoflavone (HI); middle isoflavone (MI); low isoflavone (Li); high equol (HE); middle equol (ME); low equol (LE); and oestrogen group (EG). At week 24 after DMBA was administered, tumour incidences were 60% (9/15) in CG, 13.3% (2/15) in the MI group, 13.3% (2/15) in the Li group, 6.7% (1/15) in the LE group, and 73.3% (11/15) in EG. Isoflavone/equol groups exhibited significantly lower incidences of tumour and longer mean latent period compared with CG and EG. Thiobarbituric acid reactive substances were significantly decreased in the plasma of the rats in HI and MI groups as well as in equol treatment groups compared with CG. Moreover, RT-PCR and western blotting show that the mRNA and protein expression levels of ERα and ERβ were significantly higher in isoflavone/equol treatment groups than in CG. In conclusion, isoflavone/equol intake can significantly inhibit the development of post-menopausal mammary tumours by antioxidant activities and oestrogenic activities.

Isoflavone intake inhibits the development of 7,12-dimethylbenz(a)anthracene(DMBA)-induced mammary tumors in normal and ovariectomized rats

To determine the associations between isoflavone (49.72% genistin, 5.32% daidzin, 34.54% glycitin) and breast cancer risk, 150 rats were given 5 mg 7,12-dimethylbenz(a)anthracene and half of them were ovariectomized. Then normal rats and ovariectomized rats were divided into 5 groups: control group, isoflavone high (HI), middle (MI), or low (LI) dose group consuming 100, 500, or 1000 mg isoflavones/kg diet, estrogen group (2.5 mg stilboestrol/kg diet). After 24 weeks, tumor incidences were 73% in control group, 7% in HI, 7% in MI, 27% in LI, and 80% in estrogen group for normal rats; 60% in control group, 13% in HI, 7% in MI, 13% in LI, and 73% in estrogen group for ovariectomized rats. Isoflavone treatment decreased tumor incidence and mean tumor number per rat and increased mean latent period compared with those in control group and estrogen group group significantly (p<0.05). The mRNA and protein expression of estrogen receptor β were significantly higher in isoflavone treatment groups than those in control group group. Moreover, isoflavone treatment significantly decreased 8-hydroxydeoxyguanosine content and increased superoxide dismutase level in normal rats and decreased malondialdehyde concentrations in ovariectomized rats compared with control group. In conclusions, isoflavone intake significantly inhibited the development of premenopausal and postmenopausal mammary tumors.

Withania somnifera and its emerging anti-neoplastic effects

I read with great interest the recent article by Minhas et al. (2012). Withania may exert a number of anti-proliferative effects besides its protective role in SLE. Withaferin A and withanolide are both extracted from Withinia. Of these, withaferin A is the most potent though withanolide also exhibits growth inhibiting activity (Choudhary et al. 2010). These anti-proliferative effects are clearly seen in NCI-H460 lung cancer cell lines (Yadav et al. 2010). Similarly, when applied to lung cancer cell lines, Withania augments the anti-neoplastic effects of chemotherapeutic agents such as paclitaxel (Senthilnathan et al. 2006). Withania extracts appear to have an immunomodulatory effect and enhance paclitaxel-induced inhibition of cell proliferation. Witharin also appears to be a radio sensitizer as it increases radio sensitivity of tumors and thereby enhances the apoptotic effects of radiotherapy (Muralikrishnan et al. 2010). Withania extracts appear to inhibit proliferation in HCT-15 colon cancer cell lines. For instance, it attenuates azoxymethane-induced colon cancer by modulating the levels of immunoglobulins G, A, and M and by exerting direct anti-proliferative effects (Mathur et al. 2004). 1-oxo-5beta, 6beta-epoxy-witha-2-enolide derived from Withania root has shown efficacy in the management of ultraviolet-B-induced dermatological carcinomas (Devi et al. 1996). Withania also appears to play a role in chemoprevention of skin malignancies. For instance, extracts from Withania roots have been shown to prevent the development of 7,12-dimethylbenz[a]anthracene (DMBA)induced skin cancers (Prakash et al. 2002). The above examples clearly illustrate the anti-proliferative effects of Withania and the need for further studies in this regard.

P2X7 receptors and epithelial cancers

AbstractThe P2X7A receptor is the main physiological pro‐apoptotic mechanism in epithelia in vivo. The natural ligand of the P2X7A is ATP. The P2X7A is expressed abundantly in epithelial reserve (stem) cells, and it regulates epithelial‐cell terminal differentiation. The high expression of P2X7A in reserve cells renders the cells sensitive to conditions of increased extracellular ATP, in particular upon neoplastic transformation, and to the induction of apoptosis. Cellular expression of the P2X7A is reduced in cancer cells of epithelia derived from the ectoderm, the urogenital sinus, and the distal paramesonephric duct. Low levels of P2X7A have been linked to cancer development because reserve cells harboring a defective P2X7A mechanism can escape P2X7A pro‐apoptotic control. Mechanisms that induce reduced expression of P2X7A in cancer epithelial cells involve hypermethylation of the P2X7 gene and decreased transcription; enhanced degradation of the P2X7 transcript through the action of micro‐RNAs miR‐186 and miR‐150; and epidermal growth factor (EGF) regulated, protein kinase‐A (PKA)‐mediated de‐glycosylation of the P2X7A. Reduced cellular P2X7A content has been proposed as a biomarker for human breast, bladder, ectocervix, endocervix, and endometrial cancers. The chemotherapeutic potential of augmented P2X7A‐dependent apoptosis of neoplastic epithelial cells was recently demonstrated in the mouse in vivo. Local treatment on the skin with the P2X7A‐specific agonist benzoyl‐ATP (BzATP) decreased the development of 7,12‐dimethyl‐benz(a)anthracene (DMBA)/12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced skin papillomas and skin cancers by 50%, by an effect that involves augmentation of keratinocytes reserve cells apoptosis. The P2X7A mechanism represents a novel pharmaceutical target for the prevention and treatment of epithelial neoplasia through the induction of apoptosis. WIREs Membr Transp Signal 2012,1:349–371. doi: 10.1002/wmts.33For further resources related to this article, please visit the WIREs website.

Quercetin suppresses cytochrome P450 mediated ROS generation and NFκB activation to inhibit the development of 7,12-dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch carcinomas

Increased production of reactive oxygen species (ROS) has long been recognized to play a pivotal role in carcinogenesis. Quercetin, a naturally occurring dietary flavonoid is known for its ROS scavenging properties. The present study was designed to investigate the chemopreventive and chemotherapeutic effects of quercetin based on cytochrome P450 (CYP) mediated ROS generation, ROS-induced cellular damage and activation of the NFκB signalling circuit during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Administration of quercetin inhibited the development of DMBA-induced HBP carcinomas by impairing CYP-mediated ROS production via downregulation of the expression of CYP1A1 and CYP1B1, and upregulation of antioxidant defences. Attenuation of ROS generation by quercetin in turn abrogated NFκB signalling by preventing the phosphorylation and degradation of IκB, nuclear translocation of NFκB and transactivation of its target genes associated with cell proliferation and apoptosis evasion. Thus dietary flavonoids such as quercetin that can block ROS generation and inhibit the redox regulated transcription factor NFκB, by virtue of their antioxidant potential are promising candidates for future antioxidant-based anticancer regimens.

The inhibitory effect of bamboo extract on the development of 7,12‐dimethylbenz[a]anthracene (DMBA)‐induced breast cancer

The incidence of breast cancer among women is high and increasing. This study investigated the inhibitory effect of an extract from bamboo Phyllostachys edulis on the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer in female Sprague-Dawley rats. The rats were fed with bamboo extract (BEX) supplemented diet or control diet, and treated with DMBA after 3 weeks of the dietary regime. The incidence of mammary tumors was monitored by palpation for the next 11 weeks. At the end of the experiment, blood samples were collected for total antioxidant capacities (TAC) assay and liver samples for phase II enzyme activity assays. The TAC values, total contents of phenolics and flavonoids of BEX were also measured. The results showed that BEX delayed the onset of mammary tumor by 1 week, decreased the tumor incidence by 44% and tumor multiplicity by 67%, and increased the total sulfotransferases (SULT) activity by 63%. BEX showed high levels of TAC, total phenolic and total flavonoids. However, the serum TAC values were not affected by BEX supplementation. In summary, the results indicate that BEX possesses a potent anti-breast cancer effect, and the upregulation of SULT activity, therefore estrogen metabolism may be the underlying mechanism.

In Vitro Antioxidative Potential of Lactoferrin and Black Tea Polyphenols and Protective Effects In Vivo on Carcinogen Activation, DNA Damage, Proliferation, Invasion, and Angiogenesis During Experimental Oral Carcinogenesis

The present study was designed to evaluate the in vitro antioxidant potential of bovine lactoferrin (bLF) and black tea polyphenols [Polyphenon-B (P-B)] as well as in vivo inhibitory effects on the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinomas. Antioxidant activity was screened using a panel of assays including 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), hydroxyl radical anion (OH*), superoxide anion (O2*-), and nitric oxide (NO) radical scavenging assays as well as assay for reducing power. The chemopreventive potential of bLF and P-B was assessed in the HBP model based on the modulatory effects on DMBA-induced oxidative DNA damage as well as the expression of proteins associated with carcinogen activation (CYP1A1, CYP1B1), cell proliferation [cyclin D1, proliferating cell nuclear antigen (PCNA), glutathione S-transferase pi (GST-P)], angiogenesis [vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR1)], and invasion and metastasis [matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of MMP-2 (TIMP-2)]. Both bLF and P-B showed high radical scavenging activity and reductive potential. Although administration of bLF and P-B alone suppressed DMBA-induced HBP tumors, combined administration of bLF and P-B was more effective in inhibiting HBP carcinogenesis by inhibiting oxidative DNA damage, carcinogen activation, cell proliferation, invasion, and angiogenesis. Our study suggests that the antioxidative property of bLF and P-B may be responsible for chemoprevention of HBP carcinogenesis by modulating multiple molecular targets.

Effect of milk on the 7,12-dimethylbenz[a]-anthracene-induced mammary tumor model in rat

Milk may be one of the risk factors in the development of breast cancer from epidemiological investigations. Our study investigated the hormones and main ingredients in milk and assessed the effects of milk on the development of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats by comparing differences among three groups: commercial milk (C), traditional milk (T) or water (W). During the 20-weeks experiment the C and T groups showed higher incidences of mammary tumors than the W group. After excluding potential confounding factors including fat and calcium, the C group was found to score higher than the T group in the indices of tumorigenesis. These findings suggested that DMBA-induced mammary tumors are more prevalent in milk-fed groups due in part to the contribution of estrogen and progesterone in milk.

Promoting Effects of Milk on the Development of 7,12-dimethylbenz(a)anthracene (DMBA)-induced Mammary Tumors in Rats

To assess the effect of milk on the development of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors, 48 female Sprague-Dawley rats treated with DMBA were divided into 3 groups and given 1 of 3 test solutions for 20 weeks as their drinking liquid: milk, estrone sulfate solution or tap water. The milk group showed a significantly great incidence (75%) in tumor development compared with the water group (38%) and was comparable to the estrone sulfate group (69%). Mean tumor number per rat in the milk group was significantly higher than that in the water group (p=0.009). We classified the mammary tumors into three histological types: intraductal papilloma, fibroadenoma, and adenocarcinoma. Although the percent of intraductal papilloma and fibroadenoma was almost same among the three groups, malignant tumor was found only in the milk and estrone sulfate groups. In conclusion, our results indicate that milk as well as estrone sulfate promotes the development of DMBA-induced mammary tumors in rat and could be associated with the occurrence of adenocarcinoma.

Oral artemisinin prevents and delays the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer in the rat

Artemisinin, a compound isolated from the sweet wormwood Artemisia annua L., has previously been shown to have selective toxicity towards cancer cells in vitro. In the present experiment, we studied the potential of artemisinin to prevent breast cancer development in rats treated with a single oral dose (50 mg/kg) of 7,12-dimethylbenz[a]anthracene (DMBA), known to induce multiple breast tumors. Starting from the day immediately after DMBA treatment, one group of rats was provided with a powdered rat-chow containing 0.02% artemisinin, whereas a control group was provided with plain powdered food. For 40 weeks, both groups of rats were monitored for breast tumors. Oral artemisinin significantly delayed ( P<.002) and in some animals prevented (57% of artemisinin-fed versus 96% of the controls developed tumors, P<.01) breast cancer development in the monitoring period. In addition, breast tumors in artemisinin-fed rats were significantly fewer ( P<.002) and smaller in size ( P<.05) when compared with controls. Since artemisinin is a relatively safe compound that causes no known side effects even at high oral doses, the present data indicate that artemisinin may be a potent cancer-chemoprevention agent.

Low-fat milk promotes the development of 7,12-dimethylbenz(A)anthracene (DMBA)-induced mammary tumors in rats.

Commercial cow milk contains considerable amounts of estrogens. Our study assessed the effect of commercial low-fat milk on the development of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats. Eighty 6-week-old female Sprague-Dawley rats received a single oral dose of 5 mg DMBA. Twenty-four hours later, the animals were divided into 4 groups of 20 animals each and given 1 of 4 test solutions for 20 weeks as their drinking liquid: low-fat (1%) milk (M), artificial milk (A), estrone sulfate solution (0.1 microg/ml, E), or tap water (W). The artificial milk was formulated to supply essentially the same calories as the milk. The low-fat milk contained 378 pg/ml estrone sulfate. Tumor incidence, the cumulative number of tumors and the sum of tumor diameters were higher in the M and E groups than in the A or W groups. Overall, the development of mammary tumors was in the order: M = E > A = W. Whereas the plasma 17beta-estradiol concentration in the M group was the 2nd highest after the E group, the plasma level of insulin-like growth factor (IGF-I) was significantly higher in the M group than in the other 3 groups. In conclusion, commercially available low-fat milk promotes the development of DMBA-induced mammary tumors in rats. The degree of the promotion is almost comparable to that of 0.1 microg/ml estrone sulfate. The high estrogen content in the milk may be responsible for the promotional effects, acting in concert with other hormones such as IGF-I.

Coffee enhances the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinomas

In this study, we examined the effect of roasted coffee extract on 7,12-dimethylbenz[a]anthracene (DMBA)-induced buccal pouch carcinogenesis in male Syrian hamsters using lipid peroxidation, reduced glutathione (GSH) and glutathione peroxidase (GPx) activity as biomarkers of chemoprevention. Forty male hamsters were divided into four groups of 10 animals. The right buccal pouches of the animals in Group 1 was painted with a 0.5% solution of DMBA in liquid paraffin three times a week. The animals in Group 2 painted with DMBA as in Group 1, received in addition 2 ml of 8% black coffee extract intragastrically three times a week on days alternate to DMBA application. Group 3 animals received coffee extract as in Group 2. Animals in Group 4 received neither DMBA nor coffee extract and served as control. The hamsters were sacrificed after an experimental period of 14 weeks. Biochemical measurements were carried out on tumour and normal pouch tissues. Administration of roasted coffee extract had no preventive effect on DMBA-induced oral cancer as revealed by the higher mean tumour volume and tumour burden compared to animals painted with DMBA alone. Diminished lipid peroxidation in the oral tumour tissue was accompanied by a significant increase in the levels of GSH and GPx. The results of the present study suggest that coffee exerts a tumour enhancing effect when administered during DMBA-induced hamster buccal pouch carcinogenesis.

The coffee-specific diterpenes cafestol and kahweol protect against aflatoxin B1-induced genotoxicity through a dual mechanism.

The diterpenes cafestol and kahweol (C&K) have been identified in animal models as two potentially chemoprotective agents present in green and roasted coffee beans. It has been postulated that these compounds may act as blocking agents by producing a co-ordinated modulation of multiple enzymes involved in carcinogen detoxification. In this study, we investigated the effects of C&K against the covalent binding of aflatoxin B1 (AFB1) metabolites to DNA. Male Sprague-Dawley rats were treated with increasing amounts of a mixture of C&K in the diet (0-6200 p.p.m.) for 28 and 90 days. A dose-dependent inhibition of AFB1 DNA-binding was observed using S9 and microsomal subcellular fractions from C&K-treated rat liver in an in vitro binding assay. Significant inhibition was detected at 2300 p.p.m. and maximal reduction of DNA adduct formation to nearly 50% of the control value was achieved with 6200 p.p.m. of dietary C&K. Two complementary mechanisms may account for the chemopreventive action of cafestol and kahweol against aflatoxin B1 in rats. A decrease in the expression of the rat activating cytochrome P450s (CYP2C11 and CYP3A2) was observed, as well as a strong induction of the expression of the glutathione-S-transferase (GST) subunit GST Yc2, which is known to detoxify highly the most genotoxic metabolite of AFB1. These data and the previously demonstrated effects of C&K against the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis at various tissue sites suggest the potential widespread effect of these coffee components against chemical carcinogenesis.

Changes in Selenium and Antioxidant Status during DMBA-Induced Mammary Carcinogenesis in Rats

Female weanling Sprague-Dawley rats were used to examine the changes that occurred in selenium and antioxidant status during the development of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors. Animals were fed an AIN-76 diet, modified to contain 20% fat (3:1 wt/wt, lard:corn oil) and 0.1, 0.035, 0.1, 1.0, 2.0 or 4.0 mg Se/kg diet. At wk 5, rats in groups 2-6 were administered by intragastric tube 4.32 mg of DMBA dissolved in corn oil. Control rats received corn oil only. A blood sample was removed from the tail vein and analyzed for selenium-dependent glutathione peroxidase (Se-GSHPx) and superoxide dismutase (SOD) activity at wk 5, and every 4 wk until wk 25. At the end of the experiment, rats were classified by tumor status, and each diet group was subdivided into two groups: those rats remaining free of tumors for 25 wk and those with tumors. DMBA treatment caused an initial decrease in erythrocyte SeGSHPx and SOD activity compared to untreated control rats. SeGSHPx activity in rats with tumors remained lower than controls, while SeGSHPX activity increased in rats with no tumors. These changes, however, were not associated with any changes in lipid peroxidation.

The effects of citrus limonoids on hamster buccal pouch carcinogenesis

Two citrus limonoids, limonin and nomilin, were tested for their effects on the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced buccal pouch epidermoid carcinomas. Forty-five female Syrian hamsters were divided into three equal groups. The left buccal pouches of the animals in each group were pretreated topically with two applications of dimethylsulfoxide (DMSO) (group I), a 2.5% solution of limonin dissolved in DMSO (group II) or a 2.5% solution of nomilin dissolved in DMSO (group III). After this initial treatment, 11 hamsters from each group were selected. The left buccal pouches of these animals were painted 2 or 3 times weekly with a 0.5% solution of DMBA in mineral oil. On alternate days the pouches were painted with DMSO (I), the 2.5% solution of limonin (II) or the 2.5% solution of nomilin (III). The 12 remaining hamsters were used as controls and were painted with mineral oil and DMSO (I), mineral oil and the 2.5% solution of limonin (II), or mineral oil and the 2.5% solution of nomilin (III). After 15 weeks the hamsters were killed, the pouches were excised and the tumors were counted and measured. Tumors of variable size were common in the animals treated with DMBA. However, the animals receiving topical applications of limonin exhibited a 60% reduction in tumor burden. Further comparisons between groups I and II showed that this reduction in tumor burden was due to a 20% decrease in tumor number and a 50% decrease in tumor mass. The results for group III showed that nomilin was considerably less effective as an inhibitor of DMBA-induced neoplasia.

Plasma estradiol and prolactin levels and their response to stress in two strains of rat with different sensitivities to 7,12-dimethylbenz[a]-anthracene-induced tumors

Number and development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumors are higher in Sprague-Dawley (SD) than Long-Evans (LE) rats and restraint retards tumor development in SD rats. Prolactin levels in LE hooded and SD rats were similar at ages 62 and 93 days whereas estradiol levels were considerably lower in the LE strain at the early age only. After chronic restraint of SD rats, corticosterone levels were unaffected while estradiol levels increased and prolactin levels decreased. The low levels of estradiol, indicative of delayed maturation in the LE rats at the time of DMBA exposure, might be the cause of the resistance of this strain to DMBA-induced tumors. The effects of stress might be mediated by increased levels of estradiol and decreased levels of prolactin in the stressed rats.

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Prolonged exposure to retinyl acetate (RA) in the diet inhibits the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary cancers in rats. The effectiveness of RA was examined when given 6 months after the administration of DMBA. Non-inbred female Sprague-Dawley rats with DMBA-induced mammary tumors were divided into 3 groups and treated for 4 weeks as follows: Group 1 served as controls, group 2 was ovariectomized, and group 3 received 328 mg RA/kg diet. Ovariectomy (OVX) markedly reduced both the number and size of the tumors. RA administration failed to induce any significant regression in tumor number but significantly retarded tumor growth when compared to tumor growth in group 1 controls. The levels of estradiol, progestin, and prolactin (PRL) receptors were significantly reduced after OVX, whereas only the levels of PRL receptors declined significantly after RA administration. Circulating progesterone concentrations were not affected in the RA-treated group but the plasma PRL level was significantly increased. The present studies show that if treatment with RA is delayed until 6 months after carcinogen administration, the protective effect of RA can still be observed although its effectiveness is less dramatic than when it is administered earlier.