Combination chemoprevention of rat mammary carcinogenesis by indomethacin and butylated hydroxytoluene

Indomethacin, a nonsteroidal antiinflammatory agent which inhibits prostaglandin biosynthesis, is an effective inhibitor of mammary carcinogenesis in rats. However, the activity of indomethacin as a chemopreventive agent is limited by toxicity. The present studies were conducted to determine if the toxic and anticarcinogenic effects of indomethacin can be modified by the phenolic antioxidant, butylated hydroxytoluene (BHT). Simultaneous administration of BHT resulted in a dose-related inhibition of indomethacin toxicity in female Sprague-Dawley rats, and increased the tolerable indomethacin dose from 50 to 150 mg/kg diet. When BHT (5000 mg/kg diet) and indomethacin (50 mg/kg diet) were administered in combination, no increased inhibition of 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis was observed above that attained by administration of BHT alone or indomethacin alone at those doses. However, when the indomethacin dose was increased to 100 mg/kg diet, an enhanced inhibition of carcinogenesis was attained when BHT and indomethacin were administered from 2 weeks prior to until 1 week after 7,12-dimethylbenz(a)anthracene administration. These data indicate that "combination chemoprevention" regimens can be utilized to reduce the toxicity of anticarcinogenic drugs. However, the BHT-indomethacin interaction appears to involve a functional or dispositional antagonism which limits the anticarcinogenic efficacy of increasing indomethacin dose level.     

Anticarcinogenic and hepatotoxic interactions between retinyl acetate and butylated hydroxytoluene in rats

The natural retinoid, retinyl acetate (RA), and the phenolic antioxidant, butylated hydroxytoluene (BHT), are both effective inhibitors of mammary carcinogenesis in rats. The present study was designed to determine if an increased inhibition of mammary carcinogenesis is obtained when RA and BHT are administered in combination. At age 50 days (time 0), virgin, female Sprague-Dawley rats received a single intragastric instillation of 16 mg of 7,12-dimethylbenz(a)anthracene dissolved in 1 ml sesame oil. Groups of 30 carcinogen-treated rats received Wayne Lab Chow supplemented with (per kg diet) 250 mg RA, 5000 mg BHT, or 250 mg RA plus 5000 mg BHT by the following schedule: -2 to +1 week; +1 week until the end of the experiment; -2 weeks to end; or none. Combined administration of RA plus BHT by the -2 weeks to end schedule was more effective in mammary cancer chemoprevention than was RA alone or BHT alone; the interaction of RA and BHT was additive. Similarly, administration of RA plus BHT by the -2 weeks to end protocol was more active in chemoprevention than was RA plus BHT administered either from weeks -2 to +1 or +1 week to end. Chronic exposure to RA plus BHT induced a high incidence of hepatic fibrosis and bile duct hyperplasia; these changes were not observed in controls and were seen in low incidence in animals exposed to RA only or BHT only. These data indicate that enhanced anticarcinogenic activity can be obtained through the use of "combination chemoprevention" regimens; however, chemopreventive compounds may interact not only to inhibit carcinogenesis but also to induce toxicity.     

Inhibition of binding of 2-acetylaminofluorene to DNA by butylated hydroxytoluene and butylated hydroxyanisole in vitro

Numerous studies have shown that the food antioxidants butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), under specific exposure conditions, can inhibit hepatocarcinogenesis induced by various carcinogens. The purpose of the present work was to study the biochemical mechanisms responsible for the anticarcinogenic activity of BHA and BHT using in vitro systems. The effects of BHA and BHT on the binding of 2-acetylaminofluorene (2-AAF) to DNA was determined in a microsomal system and in primary cultures of rat hepatocytes. It was found that both antioxidants reduce the binding of 2-AAF and that of N-OH-2-acetylaminofluorene (N-OH-2-AAF) to calf thymus DNA in the presence of liver microsomes. The inhibition was however more pronounced with the parent compound. Lower levels of DNA binding were also detected in hepatocytes incubated with 2-AAF along with BHA or BHT. These results suggest that phenolic antioxidants can exert anticarcinogenic activity through modulation of carcinogen interaction with DNA which may reflect on alteration in carcinogen metabolic activation.     

Modification by antioxidants and p, p'-diaminodiphenylmethane of 7, 12-dimethylbenz[a]anthracene-induced carcinogenesis of the mammary gland and ear duct in CD rats

The effects of antioxidants on mammary gland carcinogenesispretreated with 7, 12-dimethylbenz[a]anthracene (DMBA) in femaleSprague-Dawley rats were examined. The antioxidants used werebutylated hydroxyanisole (BHA), butylated hydr-oxytoluene (BHT),sodium L-ascorbate, -tocopherol, ethoxy-uin and p, p’-diaminodiphenylmethane(DDPM), which is an inhibitor of carcinogenesis in the liver,kidney and urinary bladder. Female Sprague-Dawley rats of 50days old were treated with 2.5 mg/100 g body weight of DMBA,and from 1 week later were given diet supplemented with 1% BHA,0.7% BHT, 5% sodium L-ascorbate, 1.5% -tocopherol, 0.5% ethoxyquinor 0.1% DDPM for 33 weeks and then killed. The incidences ofmammary tumors, carcinomas and fibroaden-omas in DMBA-treatedanimals were reduced by diet containing BHA or ethoxyquin. Dietcontaining BHT or DDPM inhibited the induction of only fibroadenomas.The incidence of ear duct tumors in DMBA-treated animals wasreduced by diet containing BHT, -tocopherol or ethoxyquin.     

Effect of dietary butylated hydroxytoluene on the in vivo distribution, metabolism and DNA-binding of 7,12-dimethylbenz[a]anthracene

The effect of dietary intake of butylated hydroxytoluene (BHT) (0.6%) on the in vivo distribution, metabolism and DNA-binding of intragastrically administered 7,12-dimethylbenz[a]anthracene (DMBA) was evaluated. Urinary excretion of DMBA increased, blood content of metabolized DMBA increased and blood content of non-metabolized DMBA decreased for rats fed the diet containing BHT as compared to rats fed the control diet. The binding of DMBA to both liver and mammary DNA decreased for rats fed the diet containing BHT as compared to controls. The liver activities of glutathione-S-transferase (GST), epoxide hydrolase (EH) and NAD(P)H-quinone reductase (QR) increased in response to BHT feeding. However, no increase in the mammary tissue activities of these enzymes was observed. These results suggest that the ability of dietary BHT to inhibit the initiation of DMBA-induced mammary carcinogenesis partly may be due to decreased binding of DMBA to mammary DNA. This effect of BHT is not due to an increase in mammary tissue activities of GST, EH and QR, enzymes involved in carcinogen detoxification, but may involve increased liver metabolism of DMBA to products that do not bind to DNA.     

Different modifying response of butylated hydroxyanisole, butylated hydroxytoluene, and other antioxidants in N,N-dibutylnitrosamine esophagus and forestomach carcinogenesis of rats

The modifying effects of antioxidants were examined in a carcinogenesis system after N,N-dibutylnitrosamine treatment. Male F344 rats were given 0.05% N,N-dibutylnitrosamine in their drinking water for 4 wk and then treated with basal diet containing 2% butylated hydroxyanisole (BHA), 1% butylated hydroxytoluene (BHT) with 7 ppm vitamin K, 0.8% ethoxyquin, 5% sodium L-ascorbate, 5% sodium erythorbate, or no added chemical for 32 wk. BHA enhanced forestomach carcinogenesis but did not enhance esophageal carcinogenesis. BHT enhanced esophageal carcinogenesis but did not enhance forestomach carcinogenesis. Ethoxyquin significantly enhanced esophageal tumorigenesis. Neither esophageal nor forestomach carcinogenesis was affected by the other antioxidants evaluated. BHA significantly increased DNA synthesis of the forestomach epithelium, whereas BHT tended to increase that of the esophageal epithelium. Thus, BHA and BHT showed different modifying responses in carcinogenesis of the esophagus and forestomach.     

Effects of butylated hydroxyanisole, butylated hydroxytoluene, and NaCl on gastric carcinogenesis initiated with N-methyl-N'-nitro-N-nitrosoguanidine in F344 rats

Promoting activities of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and NaCl and of combinations of these antioxidants with NaCl on gastric carcinogenesis initiated by N-methyl-N'-nitro-N-nitrosoguanidine [(MNNG) (CAS: 70-25-7; 1-methyl-3-nitro-1-nitrosoguanidine] were investigated in male inbred F344 rats. Animals, 6-week old, were given an intragastric administration of MNNG at 150 mg/kg body weight by gastric tube and 1 week later were placed on a diet containing BHA (0.5%), BHT (1.0%), NaCl (5.0%), BHA (0.5%) plus NaCl (5%), or BHT (1.0%) plus NaCl (5.0%) for 51 weeks. Control rats received no further treatment after MNNG administration. A single intragastric application of MNNG to rats induced multiple epithelial tumors of the forestomach and a few epithelial tumors of the glandular stomach after 52 weeks. Squamous cell carcinomas of the forestomach were seen in 2 of 18 effective rats (11.1%) in the control groups, and the incidences in the groups receiving the subsequent treatment were 45.0% with BHA, 15.8% with BHT, 30% with NaCl, 70% with BHA plus NaCl, and 52.9% with BHT plus NaCl. Differences in the incidences of squamous cell carcinoma between the controls and groups given BHA, BHA plus NaCl, and BHT plus NaCl were statistically significant. NaCl given alone after MNNG administration also significantly increased the incidence of papillomas in the forestomach. Incidences of glandular stomach tumors, adenomas and carcinomas were not affected by any of the subsequent treatments. No tumors of the stomach developed in the groups given BHA, BHT, and NaCl without MNNG pretreatment. Thus the present experiment revealed that BHA and NaCl but not BHT exert promoting activity on MNNG-induced forestomach carcinogenesis in rats and that, when BHA and BHT were given with NaCl, promotion was more marked, suggesting a synergistic effect on tumor promotion.     

Modulation of rat mammary carcinogenesis by indomethacin

Indomethacin, a nonsteroidal antiinflammatory agent which inhibits prostaglandin biosynthesis, has significant activity in inhibiting the growth and/or inducing the regression of transplantable tumors. The present study was designed to determine if, in addition to its chemotherapeutic effects, indomethacin also acts as a cancer chemopreventive agent. Fifty-day-old virgin female Sprague-Dawley rats were given a single intragastric dose of either 8 or 16 mg of 7,12-dimethylbenz(a)anthracene (time 0). Basal diet was supplemented with 25 or 50 mg of indomethacin per kg of diet by the following protocol: (a) -2 to +1 week; (b) +1 week to end; or (c) none. Administration of indomethacin by both protocols resulted in an inhibition of mammary tumorigenesis; however, the effect of -2 to +1 week indomethacin exposure was primarily on the induction of benign mammary tumors, while +1 week to end indomethacin administration inhibited the induction of both benign mammary tumors and mammary cancers. These data indicate that indomethacin has significant protective activity when administered either during the "early" stage (comprising the carcinogen-target cell interaction) or the "late" stage (postcarcinogen tumor development) of mammary carcinogenesis in rats. Possible mechanisms of indomethacin action include both local and systemic effects.     

Effects of butylated hydroxyanisole and butylated hydroxytoluene on 7,12-dimethylbenz[a]anthracene—DNA adduct formation in mouse embryo cell cultures

Neither butylated hydroxyanisole (BHA) nor butylated hydroxytoluene (BHT) significantly reduced overall 7,12-dimethylbenz[a]anthracene (DMBA)—DNA adduct formation in mouse embryo cell cultures. However, analysis of DMBA—DNA adducts by Servacel DHB chromatography and high-pressure liquid chromatography showed that treatment of cells with BHA, but not with BHT, resulted in a decreased contribution from the syn bay region dihydrodiol epoxide to overall binding.     

Selective induction of rat urinary bladder tumors by simultaneous administration of 3,2'-dimethyl-4-aminobiphenyl (DMAB) and butylated hydroxyanisole or butylated hydroxytoluene is associated with increased DMAB-DNA adduct formation

Modification of 3,2'-dimethyl-4-aminobiphenyl (DMAB) multi-organ carcinogenesis by simultaneous treatment with butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) was studied using young and old male F344 rats. Animals, 4 or 54 weeks old, were given DMAB (s.c. injection of 50 mg/kg body wt once a week for 10 weeks) along with BHA (2.0% in diet for 11 weeks) or BHT (1.0% in diet for 11 weeks). The experiments were terminated 55 weeks after the commencement. Combined administration of BHA or BHT with the carcinogen resulted in development of urinary bladder tumors in greater than 90% of both young and old rats thus treated, whereas no tumors were induced in animals given DMAB alone. In contrast, the appearance of preneoplastic lesions in the liver and pancreas was reduced by BHA or BHT treatment. Tumor development (less than 30% incidence) was also evident in the small and large intestines, prostate, preputial glands, skin/subcutis and ear duct, with no modification by BHA or BHT. No ageing effects were evident. The formations of DMAB-DNA adducts, evaluated by the enzyme-linked immunosorbent assay and immunohistochemical staining, correlated well with tumorigenesis in the urinary bladder, liver and pancreas. The selective enhancement of urinary bladder tumor induction by BHA and BHT appeared to be due to both increased DMAB-DNA adduct formation caused by metabolic alteration of DMAB in the liver and increased DNA synthesis in the urothelial cells.     

Modifying effects of concomitant treatment with butylated hydroxyanisole or butylated hydroxytoluene on N,N-dibutylnitrosamine-induced liver, forestomach and urinary bladder carcinogenesis in F344 male rats

The modifying effects of concomitant antioxidant treatment on N,N-dibutylnitrosamine (DBN)-induced carcinogenesis were investigated. Male F344 rats were given 0.05% DBN in their drinking water for 16 weeks, and simultaneously administered powder diet containing 2.0% butylated hydroxyanisole (BHA) or 0.7% butylated hydroxytoluene (BHT) for 16 weeks. Control animals received drinking water containing 0.05% DBN without antioxidant treatment. The final incidences of hepatocellular carcinomas were 100, 100 and 40% in the DBN plus BHA, DBN plus BHT and DBN treated groups, respectively, the difference being significant (P less than 0.001). Lung metastases were only observed in the DBN plus BHT group and DBN plus BHA group (50%, P less than 0.001; 7%, respectively). The incidence of papillary or nodular hyperplasia of the urinary bladder in the DBN plus BHA group was significantly higher than that of the control (P less than 0.05). Furthermore, esophageal carcinomas and papillomas were observed in all DBN treated groups, with no inter-group significant variation in yield. On the other hand, combination of DBN treatment with BHA or BHT significantly reduced the resultant incidences of forestomach hyperplasia. The results clearly demonstrated that concomitant administration of antioxidants, and in particular BHT, can modify DBN carcinogenesis.     

Comparison of the effect of butylated hydroxytoluene on N-nitrosomethylurea and 7,12-dimethylbenz[a]-anthracene-induced mammary tumors

The antioxidant butylated hydroxytoluene (BHT) when fed at a level of 0.3% in a defined semi-purified diet was found to decrease mammary tumor incidence in female Sprague-Dawley rats induced by 7,12-dimethylbenz[a]-anthracene (DMBA). however, no effect of BHT on tumor incidence was seen in animals consuming the same diet, under identical experimental conditions, but treated with the carcinogen nitrosomethylurea (NMU). Differences in effectiveness of BHT as a tumor inhibitor in the 2 model systems, and thoughts as to a possible mechanism of action with regard to BHT are discussed.     

Enhanced inhibition of mammary carcinogenesis by combined treatment with N-(4-hydroxyphenyl)retinamide and ovariectomy

Bilateral ovariectomy and dietary administration of the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) are both effective inhibitors of chemical carcinogenesis in the rat mammary gland. The present study was designed to determine whether an enhanced inhibitory effect is obtained with combined ovariectomy and 4-HPR administration, compared to either treatment alone. In separate experiments, 50-day-old virgin female Sprague-Dawley rats received either a single i.v. injection of 50 mg N-methyl-N-nitrosourea per kg body weight or a single intragastric dose of 20 mg 7,12-dimethylbenz(a)anthracene. The experimental design was the same in both the N-methyl-N-nitrosourea and 7,12-dimethylbenz(a)anthracene experiments: Group 1, 25 intact rats, placebo diet; Group 2, 25 intact rats, supplement of 782 mg 4-HPR per kg diet; Group 3, 50 ovariectomized rats, placebo diet; Group 4, 50 ovariectomized rats, supplement of 782 mg 4-HPR per kg diet. Feeding of the 4-HPR supplement was begun 7 days after carcinogen administration; ovariectomy was performed 7 days post-7,12-dimethylbenz(a)anthracene or 14 days post-N-methyl-N-nitrosourea. In both experiments, combined ovariectomy plus 4-HPR was significantly more active in suppressing mammary cancer induction than was either manipulation alone. 4-HPR was a more effective inhibitor of carcinogenesis in ovariectomized rats than in intact animals. These data indicate that 4-HPR is highly effective in inhibiting ovarian hormone-independent cancers and suggest that retinoid inhibition of mammary carcinogenesis does not involve an influence on ovarian hormone action.     

7,12-Dimethylbenz[a]anthracene-induced perinatal carcinogenesis and its modulation by butylated hydroxyanisole in mice

We discuss the transplacental and transmammary carcinogenicity of 7,12-dimethylbenz[a]anthracene (DMBA) in Swiss albino mice and its modulation by butylated hydroxyanisole (BHA). Transmission of the carcinogen by either route elicits the development of tumour in F1 individuals, and in either situation the incidence of tumours is dependent upon the dose of DMBA administered to gestating or lactating mothers or foster mothers. However, for a given dose of carcinogen, its transplacental carcinogenicity is much greater than its transmammary carcinogenicity. Transmammary carcinogenicity is evident in F1 progeny whether they are nursed by DMBA-exposed mothers, syngenic foster mothers (Swiss albino strain) or allogenic foster mothers (C57BL/6 strain), but the incidence of tumours is appreciably lower when allogenic females are the foster mothers. DMBA administered to females during gestation appears to remain as a residue, then to find its way through the transmammary route into normal F1 individuals being foster-nursed, and to produce tumours. We have also shown the influence of age, but not of parity, of foster mothers on DMBA-induced transmammary carcinogenesis in F1 individuals. In these experiments, BHA has a chemopreventive action against DMBA-induced transplacental and transmammary carcinogenesis in mice.     

Inhibition of chemically induced mammary carcinogenesis in rats by long-term exposure to butylated hydroxytoluene (BHT): interrelations among BHT concentration, carcinogen dose, and diet

In outbred female Sprague-Dawley rats long-term exposure to dietary butylated hydroxytoluene [3,5-di-tert-butyl-4-hydroxytoluene (BHT); CAS: 128-37-0] 1 week before carcinogen administration to termination resulted in a dose-related inhibition of mammary tumorigenesis and adrenocortical nodulogenesis. In animals fed the cereal-based NIH-07 diet and receiving a low dose (5 mg/rat) of 7,12-dimethylbenz [a] anthracene [(DBMA) CAS: 57-97-6], there was a significant overall inhibitory trend in tumor incidence observed among those receiving 300, 1,000, 3,000, and 6,000 ppm BHT. Maximal inhibition was approximately 50% at the highest concentration of BHT (6,000 ppm). The inhibitory effect of BHT on mammary tumor incidence was less pronounced when BHT was administered to rats initiated with a high carcinogen dose: At 15 mg DMBA/rat maximal inhibition was only 20% at the highest concentration of BHT (6,000 ppm). In contrast, when tumor yield was assessed in terms of latency or tumor multiplicity, the inhibitory effect of BHT was more pronounced in the groups given a high dose of DMBA than in the groups given a low dose. In animals given a low dose of DMBA (5 mg) and fed 6,000 ppm BHT in the casein-based AIN-76A diet, tumor incidence was inhibited by 50% of that of the controls; in contrast, when initiation was with a high dose of DMBA (15 mg), tumor incidence was decreased by only 28% of that of the controls. In animals fed the NIH-07 diet, DMBA-induced adrenocortical nodule formation was also inhibited in a dose-dependent fashion by BHT. At 5 mg DMBA maximal inhibition was 86% of control levels (6,000 ppm BHT); at 15 mg DMBA maximal inhibition was 66% of control levels (6,000 ppm BHT). However, when BHT was incorporated into the AIN-76A diet, its inhibitory effects on adrenocortical nodulogenesis were unexpectedly feeble and unrelated to carcinogen dose: In animals initiated with 5 mg DMBA and administered 6,000 ppm BHT, nodule incidence was decreased by only 25%, whereas in animals initiated with 15 mg DMBA, nodule incidence was decreased by 30% of that of the controls. These results indicate that while chronic exposure to dietary BHT suppressed the development of DMBA-induced mammary tumors and adrenocortical nodules, the degree of suppression depended on the dose of carcinogen administered, the level of BHT in the diet, and the parameter being measured. Diet-dependent differences in BHT action were observed with regard to DMBA-induced adrenocortical nodulogenesis but not with regard to mammary tumorigenesis.     

Anticlastogenic and Anticarcinogenic Potential of Thai Bitter Gourd Fruits

Thai bitter gourd fruits (Momordica charantia Linn., TBG) has been previously demonstrated to possessphase II detoxificating enzymes inducing properties, as well as the ability to reduce phase I carcinogen activatingenzyme activity in rat liver. In addition, it was partially inhibited 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary gland carcinogenesis in female Sprague-Dawley rats. In this study, we therefore examined theanticlastogenic and anticarcinogenic effect of TBG against clastogens, cyclophosphamide (CYP) and DMBA, inmice using the in vivo erythrocyte micronucleus assay and azoxymethane (AOM)-induced colon carcinogenesisin rats, respectively. For anticlastogenicity test, male mice were fed with modified AIN-76 diets containing6.25% and 12.5% of ground freeze-dried TBG for 2 weeks prior to administration of clastogens till the end ofexperiment. Blood samples were collected and counted for reticulocytes by using the fluorescent microscope. Foranticarcinogeicity test, male Wistar rats were fed with modified AIN-76 diets containing 5% and 10% groundfreeze-dried TBG for 2 weeks prior to, during and 1 week after the completion of AOM administration (15 mg/kgonce a week for 2 weeks). It was found that TBG at 6.25% resulted in a significant reduction in micronucleatedperipheral reticulocytes (MNRETs) induced by only CYP. Study on anticarcinogenic potential demonstratedthat rats fed with TBG diets at the concentration tested developed significantly higher incidence as well as themultiplicities of colon tumors than the control group. These results demonstrated that Thai bitter gourd fruitspossesses anticlastogenic potential against clastogen in the mouse. Interestingly, it had no preventive potentialagainst AOM-induced colon carcinogenesis in rat, rather increasing the incidence of colonic neoplasm whengiving during the initiation stage.     

Selective in vivo inhibition of rat mammary 7,12-dimethylbenz[a]anthracene-DNA adduct formation by dietary butylated hydroxytoluene

The in vivo formation of specific 7,12-dimethylbenz[a]-anthracene (DMBA)-DNA adducts in the mammary gland of the female Sprague-Dawley rat was studied in response to dietary butylated hydroxytoluene (BHT). Dietary BHT concentrations of 0.4 and 0.8% significantly inhibited total DMBA-DNA binding by 41.5 and 35.6% respectively, as compared to controls. However, the decrease in total binding associated with intake of BHT was not due to a uniform inhibition in the formation of all individual adducts. The formation of two adducts resulting from the binding of the anti-dihydrodiolepoxide of DMBA to deoxyguanosine (anti-dGuo) was significantly decreased by a combined average of 51.5% for rats fed BHT-supplemented diets as compared to controls. However, syn-derived DMBA-DNA adducts were not consistently inhibited by dietary BHT. Adduct formation resulting from the binding of the syn-dihydrodiolepoxide of DMBA to deoxyadenosine (syn-dAdo) was significantly inhibited only for rats fed a diet supplemented with 0.4% BHT. The formation of the syn-dGuo adduct was not affected by the feeding of BHT-supplemented diets. These results suggest that in vivo inhibition by BHT of mammary DNA adducts formed from the anti-diastereomer of DMBA may be an important contribution to the inhibitory effect of BHT on the initiation stage of DMBA-induced mammary carcinogenesis.     

Induction of forestomach lesions in rats by oral administrations of naturally occurring antioxidants for 4 weeks

The effects of naturally occurring antioxidants on rat forestomach epithelium were compared with those of synthetic antioxidants, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), of which the former is a known forestomach carcinogen. Groups of five F344 male rats were given diet containing BHA, BHT, gallic acid, syringic acid, sesamol, caffeic acid, chlorogenic acid, ferulic acid, eugenol or esculin for 4 weeks at a level of 0.7% for BHT or 2% for other compounds. Histological examination of the forestomach showed that BHA induced hyperplasia mainly in the prefundic region near the esophageal orifice, caffeic acid induced pronounced hyperplasia throughout the forestomach epithelium, and sesamol induced large ulcers and hyperplasia in the central region. Thus, these naturally occurring antioxidants showed different toxicities and abilities to induce hyperplasia in the rat forestomach.     

Inhibition of mammary gland tumors by short-term treatment of estradiol-3-benzoate associated with down-regulation of estrogen receptor ERalpha and ERbeta

Epidemiological evidence indicates that estrogens are one of the risk factors of breast cancer. However, there have been reports that pre-pubertal estrogen exposure is related to reduced breast cancer risk. These discrepancies made us investigate the time-point and duration of estrogen exposure. Our studies focus on the effect of estradiol-3-benzoate (EB) on the mammary gland that was exposed to carcinogens. Ninety-six female Sprague-Dawley rats were randomly divided into 6 groups. Animals at 7 weeks of age were injected with 7,12-dimethylbenz[a]anthracene (DMBA) in groups 1, 2 and 3 or N-methyl-N-nitrosourea (MNU) in groups 4, 5 and 6. One week later, the animals were subjected to sustained treatment with 0 micro g (groups 1 and 4), 30 micro g (groups 2 and 5) or 300 micro g (groups 3 and 6) of EB containing pellets for 4 weeks. All animals were sacrificed at 5 weeks or 21 weeks after carcinogen treatment, for the examination of mammary gland differentiation or mammary gland tumors, respectively. At 21 weeks after carcinogen treatment, the incidence of mammary tumors in group 2 was significantly decreased (P<0.05). EB treatment decreased the multiplicity of DMBA- or MNU-induced mammary gland tumors. At 5 weeks after carcinogen treatment, there were increased branchings of the mammary gland, and there was also a decrease of ERalpha and ERbeta in EB treatment groups. Taken together with these results, we conclude that EB has an inhibitory effect on mammary carcinogenesis, and it suggests that this inhibition may be associated with the differentiation of mammary gland and modulation of ERalpha and ERbeta.     

Dose-dependent effects of butylated hydroxyanisole, butylated hydroxytoluene and ethoxyquin for promotion of bladder carcinogenesis in N-butyl-N-(4-hydroxybutyl)nitrosamine-initiated, unilaterally ureter-ligated rats

Dose-dependent effects of 3 antioxidants, butylated hydroxyanisole (BHA, 2.0, 1.0 and 0.5%), butylated hydroxytoluene (BHT, 1, 0.5 and 0.25%) and ethoxyquin (0.5, 0.25 and 0.125%) on the development of preneoplastic lesions in the bladder of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-treated rats were investigated. Feeding of the antioxidants after pretreatment of 0.05% BBN commenced and unilateral ureteric ligation was combined at week 3 of the experiment. Surviving rats were killed at the end of week 24. BHA and BHT, but not ethoxyquin increased dose-dependently the incidence and number of preneoplastic lesions, papillary or nodular hyperplasia of the urinary bladder in rats treated with BBN. Particularly, the incidence and number of PN hyperplasia in rats treated with 2.0% BHA and 1.0% BHT were significantly higher than those of the control group. Thus, promoting activities of BHA and BHT, but not ethoxyquin for the urinary bladder were confirmed in this system of BBN-initiated, unilaterally ureter-ligated rats.