In 141 mastectomy specimens, performed for invasive or noninvasive carcinomas, histopathologic study was performed to assess the extent of nipple‐areola involvement by the tumor. In this study, patients were excluded if (1) the tumor was located beneath the areola; and (2) nipple and/or areola abnormalities were clinically present. Tumor involvement of the nipple and/or areola was found in 44 of 141 specimens (31%), with intraductal growth in 36 (82%) of 44, stromal invasion in 3 (7%), and ductal and stromal invasion in 5 (11%). Analysis of nipple‐areolar involvement with consideration of the different variables indicates that it occurred in association with tumor size, tumor‐areola distance, and histologic type. Such information provides clinically relevant guidelines in decision making for limited breast surgery.

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Tamoxifen-DNA adducts detected in the liver of mice treated with tamoxifen have not yet been identified. In the present study a new type of tamoxifen-DNA adduct, four stereoisomers of alpha-(N:(2)-deoxyguanosinyl)tamoxifen N:-oxide 3'-monophosphate (dG(3'P)-N:(2)-TAM N:-oxide) were prepared as standard DNA adducts by reacting 2'-deoxyguanosine 3'-monophosphate with trans-alpha-acetoxytamoxifen N:-oxide in addition to four stereoisomers of alpha-(N:(2)-deoxyguano- sinyl)tamoxifen 3'-monophosphate (dG(3'P)-N:(2)-TAM) that was reported previously. Liquid chromatography-electrospray ionization-mass spectrometry of the reaction products gave the most abundant ion at m/z 731 ([M - H](-)), which corresponded to dG(3'P)-N:(2)-TAM N:-oxide. The modified products digested by alkaline phosphatase corresponded to the isomers of dG-N:(2)-TAM N:-oxide whose structures were identified previously by mass spectrometry and nuclear magnetic resonance. Using these standard markers, we analyzed the hepatic DNA adducts of female DBA/2 mice treated with tamoxifen at a dosage of 120 mg/kg/day for 7 days by (32)P-post-labeling coupled with an HPLC/radioactive detector. Mixtures of eight isomers of dG(3'P)-N:(2)-TAM and dG(3'P)-N:(2)-TAM N-oxide were separated into six peaks, since each of the cis epimers were not separated under the present HPLC conditions. Nine adducts were detected in all liver samples of mice. An epimer of trans-dG(3'P)-N:(2)-TAM was detected as the principal DNA adduct at a level of 29.0 adducts/10(8) nucleotides, which accounted for 53.3% of the total tamoxifen-DNA adducts. Lesser amounts of cis-dG(3'P)-N:(2)-TAM (2.8%) were also observed. An epimer of the trans-dG(3'P)-N:(2)-TAM N:-oxide (3.9 adducts/10(8) nucleotides) was detected as the third biggest adduct (7.2% of the total). The cis-dG(3'P)-N:(2)-TAM N:-oxide (0.4 adducts/10(8) nucleotides) accounted for 0.7% of the total. Thus, dG(3'P)-N:(2)-TAM and dG(3'P)-N:(2)-TAM N:-oxide were identified in tamoxifen-treated mouse liver.

A new HPLC gradient system was developed for (32)P-postlabeling analysis to identify and quantify hepatic tamoxifen-DNA adducts of rats and mice treated with tamoxifen. Four stereoisomers of alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG(3')(P)-N(2)-TAM), alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen (dG(3')(P)-N(2)-N-desmethyl-TAM), and alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide (dG(3')(P)-N(2)-TAM N-oxide) were prepared by reacting either alpha-acetoxytamoxifen, alpha-acetoxy-N-desmethyltamoxifen or alpha-acetoxytamoxifen N-oxide with 2'-deoxyguanosine 3'-monophosphate, and used as standard markers for (32)P-postlabeling/HPLC analysis. Our HPLC gradient system can separate the above 12 nucleotide isomers as nine peaks; six peaks representing two each trans epimers (fr-1 and fr-2) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide, and three peaks representing a mixture of two cis epimers (fr-3 and fr-4) of nucleotides. Tamoxifen was given to female F344 rats and DBA/2 mice by gavage at doses of 45 mg/kg/day and 120 mg/kg/day, respectively, for 7 days. Totally 15 and 17 tamoxifen-DNA adducts were detected in rats and mice, respectively; among them 13 adducts were observed in both rats and mice. trans-dG-N(2)-TAM (fr-2) and trans-dG(3')(P)-N(2)-N-desmethyl-TAM (fr-2) were two major adducts in both animals. Except for these two adducts, trans-dG-N(2)-TAM N-oxide (fr-2) was the third abundant adduct that accounted for 6.4% of the total adducts in mice, while this accounted for only 0.3% in rats. A trans-isomer (fr-1) and cis-isomers (fr-3 and -4) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide were also detected as minor adducts in both animals except for cis-form of dG-N(2)-TAM N-oxide in rats. Although the administered dose for rats was 2.7-fold less than that for mice, the total adduct level of rats (216 adducts/10(8) nucleotides) were 3.8-fold higher than mice (56.2 adducts/10(8) nucleotides). Thus, these three types of tamoxifen adducts accounted for 95.0 and 92.5% of the total DNA adducts of the rats and mice, respectively. The formation of tamoxifen adducts primarily resulted from alpha-hydroxylation of tamoxifen.

The in vitro experiment was carried out following 32P-postlabeling analysis to determine the DNA-reactive bile acids present in the human body. The unconjugated and conjugated forms of cholic (CA), chenodeoxycholic (CDCA), deoxycholic (DCA) and lithocholic acid (LCA) were added to calf thymus DNA followed by 1 h of incubation at 37 degrees C. After the incubation the mixture was analyzed by the nuclease P1 modification of 32P-postlabeling. Among the 12 bile acids tested, our results showed that unconjugated CDCA and LCA and the glycine and taurine conjugates of LCA (g-LCA, t-LCA) were able to bind covalently with naked DNA in vitro without intervention of any catalyst. It was also shown that bile acid alone did not give any spot on TLC. These binding reactions depended on the bile acid concentration in a linear manner. The data on the extent of binding at a concentration of 0.1 mg/ml showed values of 28.5 (t-LCA), 23.7 (g-LCA), 3.47 (LCA) and 1.32 (CDCA) adducts per 10(8) nucleotides. These reactive bile acids were also incubated with COLO 205 human colon carcinoma cells and Hep G2 human hepatocellular carcinoma cells for 24 h, but no specific DNA adduct was formed. Further, when LCA or CDCA was administered into male Fischer 344 rats by gavage at a dose of 10 mg/rat every 8 h for 3 days, no specific DNA adduct was detected in their liver or colon. Covalent DNA adducts are believed to cause alteration of the primary structure of genes, which is potentially linked to carcinogenesis. Though our preliminary data failed to detect any bile acid-related DNA adducts in the cultured cells or in rats, the results may provide a basis for assuming some of these bile acids to be potential initiators of colon cancer.