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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. We used electrospray ionization-tandem mass spectrometry (ES-MS/MS) and 32P-postlabeling analyses to investigate the presence of tamoxifen-DNA adducts in human endometrial tissue., Methods: Endometrial DNA from eight tamoxifen-treated women and eight untreated women was hydrolyzed to nucleosides and assayed for (E)-alpha-(deoxyguanosin-N2-yl)-tamoxifen (dG-Tam) and (E)-alpha-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (dG-desMeTam), the two major tamoxifen-DNA adducts that have been reported to be present in humans and/or experimental animals treated with tamoxifen, using on-line sample preparation coupled with high-performance liquid chromatography (HPLC) and ES-MS/MS. The same DNA samples were assayed for the presence of dG-Tam and dG-desMeTam by (32)P-postlabeling methodology, using two different DNA digestion and labeling protocols, followed by both thin-layer chromatography and HPLC., Results: We did not detect either tamoxifen-DNA adduct by HPLC-ES-MS/MS analyses (limits of detection for dG-Tam and dG-desMeTam were two adducts per 10(9) nucleotides and two adducts per 10(8) nucleotides, respectively) or by 32P-postlabeling analyses (limit of detection for both adducts was one adduct per 10(9) nucleotides) in any of the endometrial DNA samples., Conclusion: The initiation of endometrial cancer by tamoxifen is probably not due to a genotoxic mechanism involving the formation of dG-Tam or dG-desMeTam.

The effect of an antioxidant, disulfiram (DSF), on the carcinogenicities of N-2-fluorenylacetamide (2-FAA) and N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) was examined. DSF given in a diet at a concentration of 0.9% for 1 week before and throughout the carcinogen treatment (0.1 mmol/kg 3 times a week for 4 weeks) reduced the incidence of mammary tumors induced with 2-FAA by 50% and extended the mean latency period of malignant tumors from 5 to 10 months. By contrast, DSF had no effect on mammary carcinogenesis by N-OH-2-FAA. Consistent with these results was the demonstration of the inhibitory effect of DSF on the first step of metabolic activation of 2-FAA, i.e., N-hydroxylation. N-hydroxylation of 2-FAA was significantly inhibited in hepatic microsomes of untreated and 2-FAA-treated male and female rats by DSF given orally. A similar inhibition was shown in vitro after preincubation of hepatic microsomes with DSF. Measurements of cytochrome P450 after pretreatment of rats or microsomes with the inhibition showed no appreciable changes in the hemoprotein content. It was concluded, therefore, that the inhibitory effect of DSF on N-hydroxylation of 2-FAA is accomplished through mechanism(s) other than depression of the cytochrome P450 level. Because both 2-FAA and DSF bind to cytochrome P450 producing a type I spectrum, DSF may interfere with the binding of 2-FAA and thus alter its metabolism.

Carcinogenicity of N-hydroxy-N-3-fluorenylacetamide for the mammary glands of female inbred F344 rats was examined after systemic and topical administration. The compound given ip produced a 60% mammary tumor incidence but was only marginally active after topical application. Female F344 rats did not develop mammary tumors after topical application of N-hydroxy-N-2-fluorenylacetamide, N-acetoxy-N-2-fluorenylacetamide, or N-acetoxy-N-3-fluorenylacetamide. These results contrasted with those reported earlier for female Sprague-Dawley rats and indicated differences in susceptibility of the mammary glands of these rat strains to tumor induction by these carcinogens.

The steroid hormone receptors in N-2-fluorenylacetamide (2-FAA)- and N-hydroxy-2-FAA-induced mammary adenocarcinomas in outbred Sprague-Dawley rats were analyzed. Both 8S and 4S estrogen-binding components have been detected in cytosols of these tumors following sucrose gradient sedimentation in low salt. Competitive binding analyses of this binder indicated a specificity profile expected of an estrogen receptor. Both androgen and progesterone receptors were also present in the cytosols of these mammary tumors. While the androgen receptor sedimented in the 8S region of the gradient, the progestin binder appeared only as a 4S moiety under similar conditions. The relative concentrations of these receptors (expressed in fmol/mg protein +/- SE) were: 17 beta-estradiol (28.6 +/- 4.1) greater than 5 alpha-dihydrotestosterone (8.5 +/- 2.2) greater than progesterone (5.0 +/- 1.3). The progesterone receptor was increased at least eightfold in the mammary adenocarcinomas from ovariectomized rats that were treated with diethylstilbestrol for 6 days. Binding equilibrium data indicated Ka = 1.2-1.8 X 10(9) M-1 for the above cytoplasmic hormone receptor complexes (Ka, association constant). Although cytosols prepared from lactating mammary gland contained appreciable quantities of glucocorticoid receptor, only trace amounts were found in the mammary adenocarcinoma.