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 2.04 micromol of compound in 50 microliter/gland of DMSO. Whereas all compounds including DMSO yielded combined malignant and benign mammary tumor incidences of 33-87% by week 82 after injection, 2,7-diNF produced 100 and 93% incidences significantly (P < 0.001) sooner than did DMSO, i.e. by weeks 23-49 and 18-48 after treatment of 30- and 50-day-old rats, respectively. Rats treated with 2,7-diNF and 9-oxo-2,7-diNF had significantly (P < 0.0001) and marginally (P = 0. 0536) more mammary tumors, respectively, than DMSO-treated rats. In 2,7-diNF-treated rats, the ratio of malignant to benign mammary tumors was 5.4, whereas in all other groups it was <0.5. N-OH-2-AAF, a potent tumorigen when applied to the mammary gland as a solid or in suspension, did not yield the expected tumorigenicity here. The contrasting tumorigenic potencies of 2,7-diNF and N-OH-2-AAF may have been prompted by differences in their solubilities in DMSO. Thus, the poorly soluble 2,7-diNF was slowly absorbed from the injection sites since residues (up to 0.9% of the dose injected) were recovered even after 45 weeks. The data indicate prolonged exposure of the mammary gland to 2,7-diNF and suggest that contamination of the environment with 2,7-diNF, even at low levels, poses substantial carcinogenic risk.

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. Significant decreases in the mean tumor number per rat in TAM, I3C and TAM+I3C-treated rats indicated a cooperative effect of the two compounds. In both DMBA-initiated and uninitiated rats, significant increases in the ratios of liver to body weight in I3C and TAM+I3C-treated groups coincided with I3C-dependent increases of hepatic cytochrome P450 levels and activities (1A1, 1A2 and 2B1/2). The ratios of uterus to body weight decreased with the number of treatments and the decreases effected by TAM were greater than those by I3C. The levels of circulating estrone were increased in response to I3C treatment and were greater in DMBA-initiated rats than in uninitiated rats, which may contribute to the preventive effect of I3C. Chemoprevention may be accomplished through up-regulation of apoptotic enzyme (caspase) activities in the mammary gland or mammary tumors. Treatment with TAM, I3C or TAM+I3C had no effect on caspase-3&7, caspase-6, caspase-8 and caspase-9 activities in the mammary tumors or mammary gland of tumor-bearing rats or that of uninitiated rats. In the mammary gland of DMBA-initiated tumor-free rats, however, I3C treatment increased the levels of caspase-3&7 and caspase-9 activities, suggesting an I3C-mediated protective effect. Even though I3C alone is a much less effective suppressing agent of mammary carcinogenesis than TAM, I3C in combination with TAM does not weaken but may foster the benefits of chemoprevention with TAM.

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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. The levels of N-debenzolyation of N-OH-2-FBA to N-hydroxy-N-2-fluorenamine (N-OH-2-FA), catalyzed by microsomal carboxylesterases in mammary gland and liver were similar and increased 1.5- and 1.7-fold, respectively, by ovariectomy. N-Debenzoylating activity in cytosols of both tissues appeared to be partially of microsomal origin. Mammary gland cytosol contained N-, O- and N,O-acyltransferase activities at levels 40-50% those of liver. N-Acyltransferase activity was determined via acetyl coenzyme A (AcCoA)-dependent acetylation of 2-FA and a new assay, N-OH-2-FAA-dependent acetylation of 9-oxo-2-FA. The latter activity was decreased in mammary gland by ovariectomy. Microsomal N-acyltransferase activities were <36% those of cytosols. AcCoA-dependent binding of N-OH-2-[ring-[3H]FBA to DNA, catalyzed by cytosol, was consistent with a two-step activation of N-OH-2-FBA involving esterase-catalyzed N-debenzoylation to N-OH-2-FA and its O-acyltransferase-catalyzed acetylation to the electrophilic N-acetoxy-2-FA. O-Acetyltransfer by mammary gland appeared to be rate-limiting since ovariectomy-dependent increases in N-debenzoylation did not increase binding with S9 fraction. Little or no sulfotransferase-catalyzed binding of N-OH-2-[ring-3H]FBA-derived N-OH-2-[ring-3H]FA was detected in the liver or mammary gland cytosol, respectively. The level of binding of N-OH-2-[ring-3H]FAA to DNA catalyzed by cytosolic N,O-acyltransferase was decreased approximately 23% in mammary gland and increased 1.2-fold in liver by ovariectomy. 32P-Postlabeling analyses indicated a single adduct N-(deoxyguanosin-8-yl)-2-fluorenamine in DNA of both tissues 24 h after one intraperitoneal injection of N-OH-2-FBA or N-OH-2-FAA. Respective levels were 3.6- and 5.5-fold greater in liver than mammary gland. After ovariectomy, the adduct levels from N-OH-2-FBA increased 1.8-fold in mammary gland and from N-OH-2-FAA decreased approximately 50% in both tissues. Thus, the ovariectomy-dependent changes in levels of enzymes activating N-OH-2-FBA and N-OH-2-FAA were consistent with in vivo DNA adduct levels in the target mammary gland, but not in the liver.