Identification of Tamoxifen−DNA Adducts Induced by α-Acetoxy-N-desmethyltamoxifen

Abstract

Treatment with tamoxifen increased the risk of endometrial cancers in breast cancer patients and women participating in the chemoprevention study. In our laboratory, tamoxifen-DNA adducts, including alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG-N(2)-TAM), were detected in the endometrium of women taking tamoxifen [Shibutani, S., et al. (1999) Chem. Res. Toxicol. 12, 646-653]. On the basis of recent animal studies, deoxyguanosinyl-N-desmethyltamoxifen (dG-N-desmethylTAM) adducts are also suspected to be formed in the liver. In the study presented here, we synthesized alpha-acetoxy-N-desmethyltamoxifen as a model activated metabolite of N-desmethyltamoxifen. The overall yield of alpha-acetoxy-N-desmethyltamoxifen from alpha-hydroxytamoxifen was approximately 42%. alpha-Acetoxy-N-desmethyltamoxifen was highly reactive to 2'-deoxyguanosine, as was similarly observed for tamoxifen alpha-sulfate. The two reaction products were identified as a mixture of epimers of the trans form or cis form of alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen (dG-N(2)-N-desmethylTAM) by mass and proton magnetic resonance spectroscopy. In addition, the trans and cis forms of dG 3'-monophosphate-N(2)-N-desmethylTAM were prepared as standard markers for (32)P-postlabeling/HPLC analysis. Using this technique, dG-N(2)-N-desmethylTAM adducts were detected in calf thymus DNA reacted with alpha-acetoxy-N-desmethyltamoxifen.

Glucuronidation of Active Tamoxifen Metabolites by the Human UDP Glucuronosyltransferases

“…In addition, this is the first report to examine the pure trans-and cis-endoxifen isomers independently. Using an optimized synthesis strategy based on the methodology described by Kitagawa et al (2000), large amounts of endoxifen were synthesized for these studies, with the trans-and cis-isomers separated and purified for kinetic analysis. Both isomers of endoxifen are O-glucuronidated; however, unlike 4-OH-TAM, no N-glucuronidation of endoxifen isomers was detected in our assays for either liver microsomes or individually overexpressed UGTs, including UGT1A4, suggesting that the demethylation of the electrophilic amine on the 4-OH-TAM side chain results in a lack of N-glucuronidation by UGTs.…”

FORMATION OF TAMOXIFEN-DNA ADDUCTS VIA O-SULFONATION, NOT O-ACETYLATION, OF α-HYDROXYTAMOXIFEN IN RAT AND HUMAN LIVERS

“…1) (Osborne et al, 1996;Dasaradhi and Shibutani, 1997). Similar adduct formation has been observed using ␣-sulfate and/or ␣-acetyl forms of ␣-OH-N-desTAM (Gamboa da Costa et al, 2000;Kitagawa et al, 2000) and ␣-OHTAM N-oxide (Umemoto et al, 1999). Therefore, TAM-DNA adducts are considered to be formed through O-sulfonation and/or O-acetylation of ␣-hydroxylated TAM and its metabolites.…”

“…Human arylamine N-acetyltransferase (NAT) I (2400 units/mg; enzyme units are expressed as nanomoles of acetic acid ester product formed from paraaminosalicylic acid per minute), NAT II (220 units/mg; enzyme units are expressed as nanomoles of acetic acid ester product formed from sulfamethazine per minute), and human liver cytosol were purchased from Discovery Labware, BD Biosciences (Bedford, MA). ␣-OHTAM (Shibutani et al, 2001), ␣-OH-N-desTAM (Kitagawa et al, 2000), and ␣-OHTAM N-oxide (Umemoto et al, 1999) were prepared previously. TAM ␣-sulfate and diastereoisomers (fr-1 and fr-2) of trans-forms and diastereoisomers (fr-3 and fr-4) of cis-forms of dG 3Ј -monophosphate-N 2 -tamoxifen (dG 3Јp -N 2 -TAM) were prepared as described previously (Dasaradhi and Shibutani, 1997;Shibutani et al, 1998a).…”

Identification and Quantification of Tamoxifen-DNA Adducts in the Liver of Rats and Mice

“…One of the trans-dG-N 2 -TAM N-oxides (fr-2) was shown to be the Table 1. third most abundant adduct in the liver of tamoxifentreated mice (9). We also synthesized and identified four stereoisomers of dG-N 2 -N-desmethyltamoxifen adducts by reacting R-acetoxy-N-desmethyltamoxifen with dG, dG 3′P and DNA (14). To identify minor tamoxifen-DNA adducts formed in animals, use of standard markers is required.…”

“…Preparation of Tamoxifen-dG3′P Standards. dG3′P-N 2 -TAM, R-(N 2 -deoxyguanosinyl)-N-desmethyltamoxifen 3′-monophosphate (dG3′P-N 2 -N-desmethyl-TAM) and dG3′P-N 2 -TAM N-oxide were prepared by reacting dG3′P with R-acetoxytamoxifen, R-acetoxy-N-desmethyltamoxifen or R-acetoxytamoxifen N-oxide, as reported previously (9,(14)(15)(16) (Figure 1). The trans and cis stereoisomers of each nucleotide were isolated by HPLC.…”