DNA adduct formation and mutant induction in Sprague-Dawley rats treated with tamoxifen and its derivatives.

The non-steroidal anti-estrogen tamoxifen is used as an adjunct chemotherapeutic agent for the treatment of all stages of breast cancer and more recently as a chemoprotective agent in women with elevated risk of developing breast cancer. While beneficial for the treatment of breast cancer, tamoxifen increases the risk of endometrial cancer. In addition, it has been shown to induce liver and endometrial tumors in rats. Tamoxifen is genotoxic in rat liver, as indicated by the formation of DNA adducts, through a metabolic pathway involving the alpha-hydroxylation of tamoxifen and N-desmethyltamoxifen. Since the contribution of these alpha-hydroxy metabolites of tamoxifen to the induction of endometrial tumors is presently unknown, we compared the extent of DNA adduct formation in liver and selected non-hepatic tissues of female Sprague-Dawley rats treated by gavage with tamoxifen, alpha-hydroxytamoxifen, N-desmethyltamoxifen, alpha-hydroxy-N-desmethyltamoxifen and N,N-didesmethyltamoxifen, or intraperitoneal injection with tamoxifen, alpha-hydroxytamoxifen, 3-hydroxytamoxifen and 4-hydroxytamoxifen. In addition, spleen lymphocytes from rats treated by gavage with tamoxifen or alpha-hydroxytamoxifen were assayed for the induction of mutants in the hypoxanthine phosphoribosyl transferase (Hprt) gene. The relative levels of binding in rats treated by gavage were alpha-hydroxytamoxifen > tamoxifen approximately N-desmethyltamoxifen approximately alpha-hydroxy-N-desmethyltamoxifen > N,N-didesmethyltamoxifen. In rats dosed intraperitoneally, the relative order of binding was alpha-hydroxytamoxifen > tamoxifen > 3-hydroxytamoxifen approximately 4-hydroxytamoxifen. None of the compounds resulted in an increase in DNA adducts in uterus, spleen, thymus or bone marrow DNA from rats treated by gavage or in uterus DNA from rats injected intraperitoneally. Neither tamoxifen nor alpha-hydroxytamoxifen increased the Hprt mutant frequency in spleen T-lymphocytes. These results confirm previous observations that tamoxifen is activated to a genotoxic agent in rat liver through alpha-hydroxylation, and also suggest that endometrial tumors in rats do not arise from the formation of tamoxifen-DNA adducts.

Comparison of hprt and lacI mutant frequency with DNA adduct formation in N-hydroxy-2-acetylaminofluorene-treated Big Blue rats.

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) is the proximate carcinogenic metabolite of the powerful rat liver carcinogen 2-acetylaminofluorene. In this study, transgenic Big Blue(R) rats were used to examine the relationship between in vivo mutagenicity and DNA adduct formation by N-OH-AAF in the target liver compared with that in nontarget tissues. Male rats were given one, two, or four doses of 25 mg N-OH-AAF/kg body weight by i.p. injection at 4-day intervals, and groups of treated and control rats were euthanized up to 10 weeks after beginning the dosing. Mutant frequencies were measured in the spleen lymphocyte hprt gene, and lacI mutant frequencies were determined in the liver and spleen lymphocytes. At 6 weeks after beginning the dosing, the hprt mutant frequency in spleen lymphocytes from the four-dose group was 16.5 x 10(-6) compared with 3.2 x 10(-6) in control animals. Also at 6 weeks, rats given one, two, or four doses of N-OH-AAF had lacI mutant frequencies in the liver of 97.6, 155.6, and 406.8 x 10(-6), respectively, compared with a control frequency of 25.7 x 10(-6); rats given four doses had lacI mutant frequencies in spleen lymphocytes of 55.8 x 10(-6) compared with a control frequency of 20.4 x 10(-6). Additional rats were evaluated for DNA adduct formation in the liver, spleen lymphocytes, and bone marrow by (32)P-postlabeling. Adduct analysis was conducted 1 day after one, two, and four treatments with N-OH-AAF, 5 days after one treatment, and 9 days after two treatments. N-(Deoxyguanosin-8-yl)-2-aminofluorene was the major DNA adduct identified in all the tissues examined. Adduct concentrations increased with total dose to maximum values in samples taken 1 day after two doses, and remained essentially the same after four doses. In samples taken after four doses, adduct levels were 103, 28, and 7 fmol/microg of DNA in liver, spleen lymphocytes, and bone marrow, respectively. The results indicate that the extent of both DNA adduct formation and mutant induction correlates with the organ specificity for N-OH-AAF carcinogenesis in the rat. Environ. Mol. Mutagen. 37:195-202, 2001. Published 2001 Wiley-Liss, Inc.

Characterization of the major DNA adduct formed by alpha-hydroxy-N-desmethyltamoxifen in vitro and in vivo.

Tamoxifen is hepatocarcinogenic in rats and has been associated with an increased risk of endometrial cancer in women. Recent reports suggest that it may be genotoxic in humans. N-Desmethyltamoxifen is a major tamoxifen metabolite that has been proposed to be responsible for one of the major adducts detected in liver DNA of rats treated with tamoxifen. The metabolic activation of N-desmethyltamoxifen to DNA binding products may involve oxidation to alpha-hydroxy-N-desmethyltamoxifen followed by esterification. In the study presented here, we report the synthesis of alpha-hydroxy-N-desmethyltamoxifen and the characterization of the major adduct obtained from alpha-sulfoxy-N-desmethyltamoxifen in vitro as (E)-alpha-(deoxyguanosin-N(2)-yl)-N-desmethyltamoxifen. In addition, we use (32)P-postlabeling in combination with HPLC to compare the adducts formed in the livers of female Sprague-Dawley rats treated by gavage with tamoxifen or equimolar doses of alpha-hydroxy-N-desmethyltamoxifen. We conclude that one of the major adducts formed in vivo and previously suggested to derive from N-desmethyltamoxifen is chromatographically identical to alpha-(deoxyguanosin-N(2)-yl)-N-desmethyltamoxifen.