OCTOBER 2005 VOLUME 18, NUMBER 10 © Copyright 2005 by the American Chemical Society
Letters to the Editor To the Editor: The following Letters to the Editor comment on a recent article in CRT that describes the generation of DNA adducts in human endometrial explants treated with R-hydroxytamoxifen. The issues are clear so there is no need to recapitulate them. The Editors decided to accept these Letters for publication under our new policy [(2005) Chem. Res. Toxicol. 18, 7787] because of the importance of tamoxifen as a cancer preventive agent, which is given to a large number of women who have had breast cancer. The clinical doses of tamoxifen are reported to be associated with an increase of endometrial cancer, and as the authors state, it is important to identify the mechanism of carcinogenesis to determine if it can be eliminated by structural modification. One potential mechanism is that tamoxifen acts as a genotoxic carcinogen so it is critical to determine if it induces DNA damage in human endometrial tissue. This is the focus of intense investigation by a number of laboratories including those authoring the Letters. In addition to discussing the article in CRT, the Letters summarize some key issues relevant to the measurement of DNA damage from tamoxifen. The Editors feel that these issues can and should be resolved by experiment, most directly by exchange of common tissue samples. Hopefully, the exchange of Letters will hasten this process. The Editors TX050246N 10.1021/tx050246n
Tamoxifen-DNA Adduct Formation in Human Endometrium To the Editor: We are deeply concerned about the publication in Chemical Research in Toxicology of the
paper by Kim et al. (1). In this paper, the authors describe the detection by 32P-postlabeling/HPLC analysis of DNA adducts in human endometrial explant cultures incubated with R-hydroxytamoxifen, a metabolite of tamoxifen, at concentrations of 25 and 100 µM. They conclude that this is evidence of phase II activation of the metabolite by human endometrial tissue. They further conclude that adduct formation is dependent upon Osulfation and/or O-acetylation of R-hydroxytamoxifen. Our concerns are as follows. 1. The authors fail to acknowledge that a remarkably similar experiment was published 9 years ago (2) even though they cite the paper containing it in their article (ref 46). In the earlier experiment, tamoxifen-related DNA adducts were detected in human endometrial explants treated with 20, 100, and 500 µM R-hydroxytamoxifen, using 32P-postlabeling/TLC analysis. The extent of DNA adduct formation is similar in both studies. In our view, the work by Kim et al. adds nothing new to the one previously published. Furthermore, we feel that they are remiss in not properly citing a major conclusion of the earlier study. 2. R-Hydroxytamoxifen is a very minor metabolite of tamoxifen. While plasma concentrations of tamoxifen and N-desmethyltamoxifen are approximately 1 µM, the concentrations of R-hydroxytamoxifen are at most 1% of this. For instance, Poon et al. (3) reported an estimated level of 75 pg/mL (ca. 0.2 nM) in the plasma. We question the rationale of conducting in vitro incubations with 100 µM R-hydroxytamoxifen, if plasma concentrations are several orders of magnitude lower, even allowing for shorter exposure times and the speculation that tissue concentrations of R-hydroxytamoxifen may be higher than plasma or serum concentrations. Carmichael et al. (2) discussed this limitation and concluded that at physiological levels it was unlikely that adduct formation would occur to a sufficient extent to pose a genotoxic risk. Despite citing the Poon et al. paper (ref 71) as evidence for the presence of R-hydroxytamoxifen in the plasma of tamoxifen-treated women, Kim et al. failed to acknowledge the huge discrepancies between the concentrations of R-hydroxytamoxifen used in their incubations and those found in vivo.
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3. Kim et al. do not present any data to support their claim that O-sulfation and/or O-acetylation is involved in tamoxifen-DNA adduct formation in the endometrium samples. It has been shown that R-hydroxytamoxifen has the capability to react directly with DNA (4). Although the extent of reaction is enhanced by acid catalysis, adducts can be formed at neutral pH. The extent of adduct formation by R-hydroxytamoxifen in endometrial explants is extremely low in comparison with other systems (e.g., rat hepatocytes) in which active metabolic processes have been demonstrated (5). Thus, the observation of low levels of DNA adduct formation in both endometrium studies (1, 2) is most likely a consequence of the intrinsic weak chemical reactivity of the metabolite. Without using inhibitors of O-sulfation and/or Oacetylation, Kim et al. cannot exclude the possibility that all that they are observing is the direct reaction of R-hydroxytamoxifen with DNA. In any case, R-hydroxytamoxifen is not a substrate for human acetyltransferases (6). 4. Kim et al. cite a paper by Sharma et al. (7) (ref 54) in which it was claimed that tamoxifen-DNA adducts were formed upon the incubation of tamoxifen with human endometrium explants. They fail to mention that when Carmichael et al. (2) conducted similar incubations, tamoxifen-DNA adducts were not detected. 5. Kim et al. note that when HPLC/ES-MS/MS was used to assay samples provided by Sharma and colleagues, tamoxifen-DNA adducts were not detected (8). They attribute this to the fact that only seven samples were assayed, but they fail to note that it was highly unlikely (p ) 0.02, Fisher exact test) that the failure to detect tamoxifen-DNA adducts was due to chance. 6. Kim et al. suggest that the inability of other groups to detect tamoxifen-DNA adducts is due to the lower sensitivity of the methods that they use. Dr. Shibutani did not participate in a recent interlaboratory trial (9) that would have directly tested this claim. Nevertheless, the HPLC/ES-MS/MS method has a limit of detection for tamoxifen-DNA adducts of 0.2 adducts/108 nucleotides. This is nearly 2 orders of magnitude lower than the levels reported in the Kim et al. paper. Furthermore, when endometrial DNA samples from tamoxifen-treated monkeys were assayed by their 32P-postlabeling method (10) and the HPLC/ES-MS/MS method (11), similar results were obtained (
