Inhibitory effects of a dietary phytochemical 3,3'-diindolylmethane on the phenobarbital-induced hepatic CYP mRNA expression and CYP-catalyzed reactions in female rats.

3,3'-diindolylmethane (DIM), derived from indole-3-carbinol (I3C), is used as a dietary supplement for its putative anticancer effects that include suppression of mammary tumor growth in female rats. The mechanism of action DIM may involve its interaction(s) with hepatic cytochromes P450 (CYPs) catalyzing oxidations of 17beta-estradiol (E2). Our study showed that DIM added to hepatic microsomes of female Sprague-Dawley rats was primarily a competitive inhibitor of beta-naphthoflavone (beta-NF)- or I3C-induced CYP1A1 probe activity, and a potent mixed or uncompetitive inhibitor of phenobarbital (PB)-induced CYP2B1 or CYP2B2 probe activity, respectively.

Tumorigenicity and genotoxicity of an environmental pollutant 2,7-dinitrofluorene after systemic administration at a low dose level to female rats.

Environmental pollution with nitroaromatic compounds may pose health hazards. We have examined the tumorigenicity in female Sprague-Dawley rats of 2,7-dinitrofluorene (2,7-diNF) and 9-oxo-2,7-diNF administered by intraperitoneal (i.p.

Suppression of mammary gland carcinogenesis by post-initiation treatment of rats with tamoxifen or indole-3-carbinol or their combination.

This study examined whether suppression of mammary gland carcinogenesis elicited by low doses of tamoxifen (TAM) can be enhanced by concomitant treatment of rats with indole-3-carbinol (I3C), a component of cruciferous vegetables and a dietary supplement used for its putative antiestrogenicity. Two weeks after one oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) at 65 mg/kg body weight, female Sprague-Dawley rats started treatment with TAM (10 microg/rat) by subcutaneous injection, I3C (250 mg/kg body weight) by oral gavage, TAM+I3C or their respective vehicles three times per week, for up to 20 weeks. Significant increases in the median latency of malignant mammary tumors and decreases in the mean tumor mass per rat were due to TAM.

Quantitative analysis of the oxidative DNA lesion, 2,2-diamino-4-(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone), in vitro and in vivo by isotope dilution-capillary HPLC-ESI-MS/MS.

A major DNA oxidation product, 2,2-diamino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone), can be generated either directly by oxidation of dG or as a secondary oxidation product with an intermediate of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). Site-specific mutagenesis studies indicate that oxazolone is a strongly mispairing lesion, inducing approximately 10-fold more mutations than 8-oxo-dG. While 8-oxo-dG undergoes facile further oxidation, oxazolone appears to be a stable final product of guanine oxidation, and, if formed in vivo, can potentially serve as a biomarker of DNA damage induced by oxidative stress.

Suppression of 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis by pre-initiation treatment of rats with beta-naphthoflavone coincides with decreased levels of the carcinogen-derived DNA adducts in the mammary gland.

Background: Mechanisms underlying prevention by beta-naphthoflavone (beta-NF) of mammary carcinogenesis initiated with 7,12-dimethylbenz[a]anthracene (DMBA) in the rat were elucidated.

Methods And Results: Treatment of female Sprague-Dawley rats with beta-NF at 40 mg/kg b.wt.

Electrospray ionization-tandem mass spectrometry and 32P-postlabeling analyses of tamoxifen-DNA adducts in humans.

Background: Although the nonsteroidal antiestrogen tamoxifen is used as an adjuvant chemotherapeutic agent to treat hormone-dependent breast cancer and as a chemopreventive agent in women with elevated risk of breast cancer, it has also been reported to increase the risk of endometrial cancer. Reports of low levels of tamoxifen-DNA adducts in human endometrial tissue have suggested that tamoxifen induces endometrial cancer by a genotoxic mechanism. However, these findings have been controversial.

Differences in the hepatic P450-dependent metabolism of estrogen and tamoxifen in response to treatment of rats with 3,3'-diindolylmethane and its parent compound indole-3-carbinol.

Indole-3-carbinol (I3C), present in cruciferous vegetables, and its major in vivo product 3,3'-diindolylmethane (DIM), have been reported to suppress estrogen-responsive cancers. This effect may be mediated through the modification of cytochrome P450 (CYP) complement and activities leading to estrogen detoxification. We examined the effects of a 4-day treatment of female Sprague-Dawley rats with DIM at 8.

Effects of treatment of rats with indole-3-carbinol on apoptosis in the mammary gland and mammary adenocarcinomas.

Induction of apoptosis is an approach to suppress carcinogenesis. The effects of a 12-week treatment of female Sprague-Dawley rats with indole-3-carbinol (I3C), beta-naphthoflavone or vehicle (40% ethanol in corn oil), by oral gavages starting 3 weeks after initiation of mammary tumorigenesis with 7,12-dimethylbenz[alpha]anthracene, on apoptotic activities in the mammary adenocarcinomas were examined. Apoptotic cells in tumor sections were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and quantitated by light microscopy and an Image-Plus Program.

Modulations of P450 mRNA in liver and mammary gland and P450 activities and metabolism of estrogen in liver by treatment of rats with indole-3-carbinol.

Indole-3-carbinol (I3C), found in cruciferous vegetables, has been shown to suppress tumorigenesis at estrogen-responsive sites. This effect may be mediated through modification by I3C of the cytochrome P450 (CYP) complement and activities leading to estrogen detoxication. In this study, we examined the effects of 4- and 10-day treatments of female Sprague-Dawley rats with I3C at 5, 25, and 250 mg/kg body weight, administered by oral gavage, on CYP mRNA expression in the liver and mammary gland, CYP-dependent activities, and the metabolism of 17beta-estradiol (E2) and estrone (E1) by liver microsomes.

DNA adducts from nitroreduction of 2,7-dinitrofluorene, a mammary gland carcinogen, catalyzed by rat liver or mammary gland cytosol.

Nitrofluorenes are mutagenic and carcinogenic environmental pollutants arising chiefly from combustion of fossil fuels. Nitro aromatic compounds undergo nitroreduction to N-hydroxy arylamines that bind to DNA directly or after O-esterification. This study analyzes the DNA binding and adducts from the in vitro nitroreduction of 2,7-dinitrofluorene (2,7-diNF), a potent mammary carcinogen in the rat.

Oxidations of 17beta-estradiol and estrone and their interconversions catalyzed by liver, mammary gland and mammary tumor after acute and chronic treatment of rats with indole-3-carbinol or beta-naphthoflavone.

Altered cytochrome P450-catalyzed metabolism of 17beta-estradiol (E2) and estrone (E1) in the liver and (or) extrahepatic tissues may affect estrogen-sensitive tumorigenesis. We examined the effects of oral treatments of (i) indole-3-carbinol (13C) at 250 or 500 mg/kg or beta-naphthoflavone (beta-NF) at 40 mg/kg of body weight (bw)/day from 51 to 54 days of age (acute regimen), and (ii) 13C at 250 mg/kg or beta-NF at 20 mg/kg bw given 3x/week from 10 to 22 weeks of age (chronic regimen) in female Sprague-Dawley rats. We determined the effects of these treatments on the P450 content and P450 (CYP)-specific activities in the liver, P450-dependent metabolism of E2 and E1 by the liver and mammary gland, and interconversion of E1 and E2 catalyzed by 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in these tissues and malignant mammary tumors.

Post-initiation treatment of rats with indole-3-carbinol or beta-naphthoflavone does not suppress 7, 12-dimethylbenz[a]anthracene-induced mammary gland carcinogenesis.

Indole-3-carbinol (I3C) and beta-naphthoflavone (beta-NF), blocking agents of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mammary gland carcinogenesis, were examined as potential post-initiation suppressing agents. Treatment of female Sprague-Dawley rats with I3C (250 mg/kg body weight (b.w.

Reductions of nitro and 9-Oxo groups of environmental nitrofluorenes by the rat mammary gland in vitro.

Nitrofluorenes and C-9-oxidized nitrofluorenes are widespread environmental genotoxins which may be relevant for breast cancer on the basis of their carcinogenicities, particularly of 2, 7-dinitrofluorene (2,7-diNF), for the rat mammary gland. Since their metabolism to active carcinogens may involve nitroreduction, this study examined the reduction of 2-nitrofluorene (2-NF) and 2,7-diNF and their 9-oxo- and 9-hydroxy (OH) derivatives by the rat mammary gland. Cytosolic fractions catalyze NADH- and NADPH-dependent reductions of the 2-nitro and 9-oxo to the respective 2-amino and 9-OH compounds at rates 4- and >/=10-fold greater than those with microsomes.

A novel 4 S [3H]beta-naphthoflavone-binding protein in liver cytosol of female Sprague-Dawley rats treated with aryl hydrocarbon receptor agonists.

beta-Naphthoflavone (beta-NF) is a widely used inducer of phase-I and phase-II enzymes controlled by aryl hydrocarbon receptor (AhR). Studies of competitive binding with (3)H-labelled 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC) and benzo[a]pyrene (B[a]P) have shown that beta-NF is a high-affinity ligand for AhR and also for polycyclic aromatic hydrocarbon (PAH)-binding protein, both soluble proteins of rat liver in 8 S and 4 S fractions, respectively, of sucrose gradients. This study examined binding of [(3)H]beta-NF to liver cytosolic proteins of female Sprague-Dawley rats.

Potent carcinogenicity of 2,7-dinitrofluorene, an environmental pollutant, for the mammary gland of female Sprague-Dawley rats.

Nitrofluorene compounds are environmental pollutants chiefly from incomplete combustion. This study examined carcinogenicities after one intramammary injection of 2-nitrofluorene (2-NF), 2, 7-dinitrofluorene (2,7-diNF) or dimethyl sulfoxide (DMSO) (solvent control) to 30-day-old and of 2-NF, 9-OH-2-NF, 9-oxo-2-NF, 2,7-diNF, 9-oxo-2,7-diNF, 2,5-dinitrofluorene, 9-oxo-2,4,7-trinitrofluorene, N-OH-2-acetylaminofluorene (N-OH-2-AAF) (carcinogen control) or DMSO to 50-day-old female Sprague-Dawley rats. In 30- and 50-day-old rats 6 and 8 glands/rat, respectively, were injected with 2.

Nitroreduction of nitrated and C-9 oxidized fluorenes in vitro.

Widespread environmental pollution with mutagenic and carcinogenic nitrofluorenes contributes to human health risks. Since nitroreduction leads to activation of many nitro compounds, nitroreduction of the nitrofluorene (NF) derivatives by one- and two-electron reductants was examined. Rates of nitroreduction catalyzed by xanthine oxidase (XO)/hypoxanthine and measured via stimulation of acetylated cytochrome c reduction increased with the number of nitro groups and oxidation at C-9: 9-oxo-2,4,7-triNF > 9-oxo-2,7-diNF > 2,7-diNF > 9-oxo-2-NF = 2,5-diNF > 9-hydroxy-2-NF > 2-NF.

Modulation by beta-naphthoflavone of ovarian hormone dependent responses in rat uterus and liver in vivo.

The potential of an aryl hydrocarbon receptor (AhR) agonist, beta-naphthoflavone (beta-NF), to modulate ovarian hormone responses in the uterus and liver of 50-day-old Sprague-Dawley rats was examined. Treatment with beta-NF at 40 mg/kg of body weight consisted of 3 or 9 intraperitoneal injections in corn oil administered to ovariectomized (OVX) and sham-treated (SH) rats on day 5 through 7 or 1 through 9 after surgery performed on day 42 or 40 of age, respectively. Treatment of SH rats with either dose regimen of beta-NF effected a decrease (approximately 80%) in the uterine peroxidase activity, which was similar to that effected by ovariectomy (> 93%).

Aryl sulfotransferase IV deficiency in rat liver carcinogenesis initiated with diethylnitrosamine and promoted with N-2-fluorenylacetamide or its C-9-oxidized metabolites.

Down regulation of aryl sulfotransferase IV (AST IV) in promotion/progression of liver carcinogenesis by N-2-fluorenylacetamide (2-FAA) has been established. This study examined whether the C-9 oxidized metabolites of 2-FAA, which have recently been shown to promote diethylnitrosamine (DEN)-initiated liver carcinogenesis in male Sprague-Dawley rats, effect the above change. Hence, in DEN-initiated rats, the effects of promoting regimens of 9-OH-2-FAA or 9-oxo-2-FAA, 15 oral doses at 50 and 100 mumol/kg of body weight, were compared to those of 2-FAA at 50 mumol/kg of body weight and of the vehicle on the activity of N-hydroxy(OH)-2-FAA sulfotransferase (ST), an isozyme of AST IV and AST IV expression and distribution.

Effect of ovariectomy on the in vitro and in vivo activation of carcinogenic N-2-fluorenylhydroxamic acids by rat mammary gland and liver.

N-Hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) and its benzamide analogue N-OH-2-FBA are mammary gland carcinogens in the female Sprague-Dawley rat. Ovariectomy inhibits tumorigenicity of topically applied N-OH-2-FAA suggesting modulation of carcinogen-activating enzymes in the gland. This study concerned the activation of N-OH-2-FAA and N-OH-2-FBA by the mammary gland and liver, a chief site of metabolism, from 50-day-old female rats and effects on the activation of ovariectomy performed at 22 days of age.

Debenzoylating and deacetylating activities of rat liver and mammary gland microsomes. Effect of ovariectomy.

This study compared the rates of N-deacylations of N-hydroxy-N-2-fluorenylbenzamide (N-OH-2-FBA) with those of its analogue, N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA), by the mammary gland (tumor target for both compounds) and the liver of female Sprague-Dawley rats and examined the effect of ovariectomy on these activities. N-Debenzoylation of N-OH-2-FBA was catalyzed by the mammary and liver microsomes of 50-day-old female rats at similar rates (approximately 24 nmol/min/mg). The activity of both tissues increased (up to 1.

Detection of N-(deoxyguanosin-8-yl)-2-fluorenamine in DNA of peritoneal serosa and liver after intraperitoneal exposure of rats to N-hydroxy-N-2-fluorenylbenzamide or N-hydroxy-N-2-fluorenylacetamide.

DNA adduct formation was examined in rat peritoneal serosa, a tumor target for i.p. administered aqueous suspensions of N-hydroxy-N-2-fluorenylbenzamide (N-OH-2-FBA) and N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA), and compared to that in the liver, which is a tumor target for N-OH-2-FAA in the male rat.

Peroxidative metabolism of carcinogenic N-arylhydroxamic acids: implications for tumorigenesis.

Peroxidative oxidations of chemical carcinogens including N-substituted aryl compounds could result in their metabolic activation because the products react with cellular molecules and lead to cytotoxicity, mutagenicity, and carcinogenicity. In vivo, peroxidative activities are chiefly of neutrophilic leukocyte origin. Neutrophils may be attracted to the site(s) of exposure to carcinogen and, via phagocytosis and respiratory burst, release oxidants that catalyze carcinogen activation and/or cause DNA damage.

Modulations of hepatic gamma-glutamyltranspeptidase and N-hydroxy-N-2-fluorenylacetamide sulfotransferase activities following treatment of rats with a hepatocarcinogenic regimen: effect of partial hepatectomy.

The feasibility of using biochemical assays of gamma-glutamyltranspeptidase (gamma-GTP) and N-hydroxy-N-2-fluorenylacetamide sulfotransferase (N-OH-2-FAA ST) activities to monitor the effects of treatment of male Sprague-Dawley rats with a two-stage hepatocarcinogenic regimen was investigated. One week after initiation with diethylnitrosamine (200 mg/kg of bw), the rats were treated with 10 oral doses within 2 weeks of N-2-fluorenylacetamide (2-FAA) at 0.05 mmole/kg or vehicle (corn oil) at 5 ml/kg of body weight.

Activation of the carcinogens N-hydroxy-N-2-fluorenylbenzamide and N-hydroxy-N-2-fluorenylacetamide via deacylations and acetyl transfers by rat peritoneal serosa and liver.

Intraperitoneally administered N-hydroxy-N-2-fluorenylbenzamide (N-OH-2-FBA) and N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) are carcinogenic for rat peritoneum. The potential of peritoneal serosa to activate these compounds via deacylations and acyl transfers was compared to that of liver. N-Deacylations of N-OH-2-FBA and N-OH-2-FAA to N-2-fluorenylhydroxylamine (N-OH-2-FA) were faster by liver than serosa and by microsomes than cytosols.

Metabolism of the carcinogen N-hydroxy-N-2-fluorenylacetamide by rat peritoneal neutrophils.

The in vitro metabolism of a locally carcinogenic N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) by rat peritoneal polymorphonuclear leukocytes (PMNL), chiefly neutrophils, elicited with intraperitoneal injections of proteose peptone, was examined. At 10(6) PMNL/ml in media containing halide (X-), 0.14 M Cl- +/- 0.