Elsevier

Toxicology

Volume 249, Issue 1, 10 July 2008, Pages 85-90
Toxicology

Short communication
Quantifiable mRNA transcripts for tamoxifen-metabolising enzymes in human endometrium

https://doi.org/10.1016/j.tox.2008.04.009Get rights and content

Abstract

Tamoxifen has been used in the management of receptor-positive breast cancer for >20 years. Usage confers an elevated risk of developing endometrial carcinoma. Its mechanism of carcinogenicity remains unresolved with controversy as to whether or not this is mediated through a genotoxic mechanism. Usage is not only associated with an elevated occurrence of endometrioid endometrial carcinoma, but also type 2 and mixed epithelial-stromal tumours (MESTs) that have a poorer prognosis. Following hysterectomy, endometrial tissues (n = 18) classified as benign (n = 6), non-tamoxifen-associated carcinoma (n = 6) and tamoxifen-associated carcinoma (n = 6) were obtained; quantitative gene expression was performed. Employing real-time RT-PCR, the relative gene expressions of phase I/II metabolic enzymes CYP1A2, CYP1B1 and CYP3A4, cathechol-O-methyltransferase (COMT) and SULT2A1 were ascertained. Measurable mRNA transcripts, especially for those genes associated with tamoxifen bioactivation, were quantifiable in all the tissues examined. Whether this is evidence that generation of genotoxic tamoxifen metabolites may occur in human endometrial tissue remains to be ascertained.

Introduction

Usage of tamoxifen, a selective oestrogen receptor modulator, in the management of oestrogen receptor-positive and/or progesterone-positive breast cancer is established (Powles, 1997). It is also administered for chemoprevention in high-risk women (Cuzick et al., 2003, Fisher et al., 2005). Of some 40,000 UK-based women diagnosed with breast cancer every year, 75% are amenable to tamoxifen treatment (Kendall et al., 2007). Tamoxifen usage is associated with an elevated risk of endometrial carcinoma (Fisher et al., 1994, van Leeuwen et al., 1994, Rutqvist et al., 1995, Curtis et al., 1996) and the mechanism remains unclear (Singh et al., 2007). In the livers of treated rats, tamoxifen gives rise to DNA adducts via a two-phase metabolism (Han and Liehr, 1992, Osborne et al., 1996, Tannenbaum, 1997); it is a potent hepatocarcinogen in this species (Greaves et al., 1993, Williams et al., 1993). Phase I metabolism results in two major metabolites, N-desmethyltamoxifen and 4-hydroxytamoxifen (Phillips, 2001, Boocock et al., 2002). Although the relatively minor route, oxidative metabolism of the ethyl side chain may generate the more potent electrophilic species, α-hydroxytamoxifen (Osborne et al., 1999, Phillips et al., 1999). Cytochrome P450 isoforms, including CYP3A4, CYP1A2 and CYP1B1, generate such phase I metabolites (Boocock et al., 2002, Sharma et al., 2003).

Phase II generation of the ultimate carcinogen of tamoxifen is via sulfate ester formation from α-hydroxytamoxifen catalysed by hydroxysteroid sulfotransferase (ST2A2 in rat; Phillips et al., 2005, SULT2A1 in human; Apak and Duffel, 2004). In rat hepatocytes, tamoxifen–DNA adduct formation is reduced both by co-incubation with dehydroisoandrostene-3-sulfate, a sulfotransferase (SULT) inhibitor, and by incubation in sulfate-free media (Davis et al., 1998). The sulfate esters generate carbocations that in turn react with exocyclic amino groups of guanine or adenine in DNA (Phillips et al., 2005). Whether such metabolism occurs in human tissues is unknown. It is also unclear to what extent the human SULT isoforms might activate α-hydroxytamoxifen (Phillips, 2001); reports dispute the presence (Shibutani et al., 1999) or absence (Carmichael et al., 1999, Beland et al., 2004) of tamoxifen–DNA adducts in human endometrium.

Elevated risk of endometrial carcinoma appears to be highest after long-term use (MacMahon, 1997, Bernstein et al., 1999, Bergman et al., 2000, Swerdlow and Jones, 2005). Long-term therapy is associated with a more common presentation of mixed epithelial-stromal tumours (MESTs), tumours that are p53-positive and oestrogen-independent, in contrast to oestrogen-driven, p53-negative type 1 disease more common in non-tamoxifen-associated cases (Bergman et al., 2000, Curtis et al., 2004, Swerdlow and Jones, 2005). Elevated risk remains even after cessation of tamoxifen use (Mignotte et al., 1998, Bernstein et al., 1999). Type 1 tamoxifen-associated endometrioid carcinoma predominantly occurs within the first 3 years of treatment onset (Magriples et al., 1993, Schlesinger et al., 1998). After prolonged use or cessation of therapy, there is an elevation of type 2 endometrial carcinomas (Dallenbach-Hellweg et al., 2000, Singh et al., 2007). Our study set out to examine whether there is evidence of gene expression in human endometrium for the main phase I or phase II enzymes associated with tamoxifen bioactivation.

Section snippets

Study participants

Study participants were patients undergoing (a) total abdominal hysterectomy for endometrial carcinoma or (b) total abdominal hysterectomy for benign disease (Table 1). Informed consent was obtained (LREC no. 05/Q1302/83; Preston, Chorley and South Ribble Ethical Committee). Tissue sets consisting of histologically confirmed tumour-adjacent (TA) or tumour (TUM) tissue were obtained and coded chronologically (N1–N6, benign; C1–C6, non-tamoxifen-associated endometrial carcinoma; T1–T6,

Results and discussion

Quantitative real-time RT-PCR analyses were carried out without prior knowledge of the histopathological findings. Averaged threshold cycle (CT) values of amplified cDNA for CYP1A2 were 29.2–31.7 for benign or TA tissue and 27.8–33.6 for TUM tissue, for CYP1B1 they were 23.1–31.3 for benign or TA tissue and 24.2 to non-detectable for TUM tissue, for CYP3A4 they were 29.5–40.0 for benign or TA tissue and 30.1–39.4 for TUM tissue, for COMT they were 21.3–30.4 for benign or TA tissue and 22.3–32.5

Acknowledgement

This work was funded by Rosemere Cancer Foundation.

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