Formation of Tamoxifen-dna Adducts via O-sulfonation, Not O-acetylation, of -hydroxytamoxifen in Rat and Human Livers

Tamoxifen (TAM) is used as the standard endocrine therapy for breast cancer patients and as a chemopreventive agent for women at high risk for this disease. Unfortunately, treatment of TAM increases the incidence of endometrial cancer; this may be due to the genotoxic damage induced by TAM metabolites. Formation of TAM-DNA adducts in rat liver correlates with the development of hepatocarcinoma. TAM-DNA adducts are proposed to be formed through Osulfonation and/or O-acetylation of -hydroxylated TAM and its metabolites. However, the role of O-sulfonation and O-acetylation in the formation of TAM-DNA adducts has not been extensively investigated. Rat or human hydroxysteroid sulfotransferases (HST), acetyltransferases, and liver cytosol were incubated with calf thymus DNA, -OHTAM, and either 3 -phosphoadenosine 5 -phosphosulfate (PAPS) or acetyl coenzyme A (acetyl-CoA) as a cofactor and analyzed for TAM-DNA adduct formation, using P postlableling/polyacrylamide gel electrophoresis analysis. TAM-DNA adduct was formed when PAPS, not acetyl-CoA, was used. No TAM-DNA adducts were produced using human N-acetyltransferase I and II. HST antibody inhibited approximately 90% of TAM-DNA adduct formation generated by the cytosol or HST, suggesting that HST is primarily involved in the formation of TAM-DNA adducts. The formation of TAM-DNA adducts with rat liver cytosol and HST was much higher than that of human liver cytosol and HST. Our results indicate that TAM-DNA adducts are formed via O-sulfonation, not O-acetylation, of -hydroxylated TAM and its metabolites. Tamoxifen (TAM) has been widely used for breast cancer therapy and also used as a chemopreventive agent for healthy women at high risk for this disease (Fisher et al., 1998; Osborne, 1998). Besides the significant benefit, long-term TAM treatment to women increases the risk of developing endometrial cancer (van Leeuwen et al., 1994; Fisher et al., 1998). TAM was listed in 1996 as a human carcinogen by the International Agency of Research on Cancer (IARC, 1996). The cellular mechanism underlying TAM-induced carcinogenesis may be due to its partial estrogenic effect through the estrogen receptors and/or genotoxic damage (reviewed by Kim et al., 2004). Actually, a high level of TAM-DNA adducts produced in the liver of rats (Han and Liehr, 1992; Osborne et al., 1996) initiates the development of hepatocellular carcinomas (Hard et al., 1993). TAM-DNA adducts detected in the endometrium of women treated with TAM (Shibutani et al., 2000; Martin et al., 2003) may also be involved in the development of endometrial cancer. Several phase I and phase II enzymes are involved in the metabolism of TAM. Cytochrome P450 converts TAM to -hydroxytamoxifen ( -OHTAM), N-desmethyltamoxifen (N-desTAM), and 4-hydroxytamoxifen (4-OHTAM) (Fig. 1) (reviewed by Kim et al., 2004). Tamoxifen N-oxide (TAM N-oxide) is produced from TAM by flavincontaining monooxygenase. -Hydroxylated forms of TAM and its metabolites are further metabolized by phase II enzymes and react with cellular DNA, resulting in the formation of TAM-DNA adducts (Phillips et al., 1994; Dasaradhi and Shibutani, 1997). In general, sulfation effectively decreases the toxicity of xenobiotics; however, in some cases, sulfation increases the toxicity. Because certain sulfate conjugates are unstable, they can form potent electrophilic species. Like the sulfate esters, acetoxy esters generated by acetylation can also be highly reactive electrophilic species. In fact, synthetic TAM -sulfate and -acetoxyTAM react rapidly with DNA, resulting in the formation of four diastereoisomers [two trans-forms (fr-1 and 2) and two cis-forms (fr-3 and 4)] of -(N-deoxyguanosinyl)tamoxifen (dG-N-TAM) adducts (Fig. 1) (Osborne et al., 1996; Dasaradhi and Shibutani, 1997). Similar adduct formation has been This research was supported by Grants ES09418 (to S.S.) from the National Institute of Environmental Health Sciences and CA38683 (to M.W.D.) from the National Cancer Institute. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.105.005330. ABBREVIATIONS: TAM, tamoxifen; -OHTAM, -hydroxytamoxifen; fr, fraction(s); dG-N-TAM, -(N-deoxyguanosinyl)tamoxifen; -OH-NdesTAM, -hydroxy-N-desmethyltamoxifen; -OHTAM N-oxide, -hydroxytamoxifen N-oxide; dG-N-N-desTAM, -(N-deoxyguanosinyl)-Ndesmethyltamoxifen; HST, hydroxysteroid sulfotransferase(s); dG3 p-N -TAM, dG3 p-monophosphate-N -tamoxifen; PAPS, 3 -phosphoadenosine 5 -phosphosulfate; acetyl-CoA, acetyl coenzyme A; NAT, N-acetyltransferase; DHEA, dehydroepiandrosterone; PAGE, polyacrylamide gel electrophoresis; HPLC, high-performance liquid chromatography. 0090-9556/05/3311-1673–1678$20.00 DRUG METABOLISM AND DISPOSITION Vol. 33, No. 11 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 5330/3059590 DMD 33:1673–1678, 2005 Printed in U.S.A. 1673 at A PE T Jornals on O cber 3, 2017 dm d.aspurnals.org D ow nladed from