Tamoxifen (Nolvadex) is a nonsteroidal antiestrogen that has become the agent of choice in the medical management of breast cancer.[1-3] Following its introduction in the 1970s for the treatment of metastatic breast cancer in postmenopausal women, the therapeutic role of tamoxifen(Drug information on tamoxifen) has grown to include initial endocrine therapy for estrogen receptor (ER)-positive disseminated breast cancer in premenopausal women, systemic adjuvant for early breast cancer in both premenopausal and postmenopausal women, and therapeutic agent for the treatment of metastatic breast cancer in men.[1-3] In patients with advanced breast cancer, complete and partial responses have been observed in 30% to 40% of unselected patients, 50% of patients with ER-positive tumors, and 60% to 70% of patients with both ER and progesterone(Drug information on progesterone) receptor (PR)-positive tumors.[1-3]
Tamoxifen has been consistently associated with improved disease-free survival and overall survival in patients with early breast cancer in a number of adjuvant therapy trials.[2-5] In addition, systemic adjuvant therapy with tamoxifen has been demonstrated to reduce the risk of developing contralateral breast cancer by 39% to 40%.[2,3] A trend toward decreased incidence of non-breast second primary malignancies in women receiving adjuvant tamoxifen therapy for early stage breast cancer has also been observed. Other benefits associated with tamoxifen include a possible cardioprotective estrogen-like effect, as well as a positive effect on bone mineral density, in postmenopausal women.[3,6,7] In addition, tamoxifen has eliminated the need for, and morbidity associated with, such surgical procedures as hypophysectomy, adrenalectomy, and possibly oophorectomy in premenopausal women with breast cancer.
Recently, several clinical trials were initiated to evaluate tamoxifen as a preventive agent in women at high risk for breast cancer.[2,8,9] Only preliminary data are currently available from these ongoing trials.
Clinical observations have suggested that tamoxifen administration is associated with a slightly increased risk of endometrial carcinoma, with a rate of detection of 0.2% to 0.3% per year compared with 0.1% in breast cancer patients not receiving tamoxifen. [10,11] The increases in endometrial cancer are similar to those observed with hormone replacement therapy. Tamoxifen therapy does not appear to be associated with an increased risk for the development of high-grade endometrial cancers with a poor prognosis, and the general consensus of the medical community is that in patients with breast cancer, the clinical benefits of tamoxifen outweigh the risk of endometrial carcinoma.[2,10] However, the risk of tamoxifen administration to healthy women without breast cancer has generated some concern in the medical community.
The debate about the use of tamoxifen as a chemoprotective agent has been fueled by animal and laboratory data demonstrating an association of tamoxifen administration with the formation of DNA adducts[12-20] and hepatocellular carcinomas in rats. In contrast to the rat data, epidemiologic observations in humans do not suggest an increased risk for liver tumors in patients treated with tamoxifen. This review summarizes human- and animal-derived laboratory data related to the hepatocarcinogenic potential of tamoxifen therapy and discusses the clinical relevance of these findings, including the formation of tamoxifen-specific DNA adducts.
Carcinogenesis is known to be a multistage process involving multiple genetic alterations in key genes involved in cell regulation and growth.  The simplest model of carcinogenesis involves two stages: initiation and promotion. Initiation happens when a mutation occurs in one of these critical genes that results in the initiated cell having a growth advantage over its normal counterpart. Promotion is best described as the clonal expansion of an initiated cell. In most cases, this model is overly simplistic; the process actually repeats itself two or more times. The clonal expansion of the initiated cell population increases the probability of a second mutation, and this often leads to further clonal expansion of its progeny. As these additional genetic alterations occur, a lesion progresses from an initiated cell to preneoplastic lesion to benign tumor to malignant neoplasm to metastasis.
One of the most common causes of genetic mutations is the binding of electrophilic chemicals to nucleic acid bases that make up DNA.[23,24] These altered nucleotides are referred to as DNA adducts. The electrophiles may be present due to exposure to environmental chemicals such as those in tobacco smoke or drugs, or even endogenous chemicals that result from normal cellular metabolism. For example, the normal steady-state level of the DNA adduct 8-hydroxydeoxyguanosine in the rat is about one adduct per 105 bases or 90,000 adducts per cell. This amount of oxidative damage has been theorized to be a major factor in the aging process and age-associated diseases such as cancer.
The effect of DNA adduct formation is lessened by DNA repair that occurs through a complex set of pathways and that can restore damaged DNA to its original normal state. Ultimately, the number of DNA adducts present in a cell at the time that a cell undergoes DNA replication for cell division is ultimately associated with the probability of mutation, with the chance of mutation increasing as the number of DNA adducts per cell increases.[23,24]
Species Differences in Metabolism
Various laboratory studies have demonstrated that tamoxifen can be biotransformed in the liver to a number of metabolites that could potentially lead to the formation of DNA adducts; the metabolites include alpha-hydroxytamoxifen, the 3,4-epoxide and 3',4'-epoxide metabolites of tamoxifen, and 4-hydroxytamoxifen (through conversion to 4-hydroxytamoxifen methide quinone).[13-19] In rat hepatocytes, alpha-hydroxytamoxifen has been demonstrated to have a high DNA-binding affinity, 25- to 49-fold greater than that of its parent compound.
Tamoxifen is metabolized more slowly by human microsomes than by those of rats, and both quantitative and qualitative differences in the formation of tamoxifen metabolites exist among rats, mice, and humans (Table 1).[25-27] For example, the concentration of alpha-hydroxytamoxifen produced by hepatocytes incubated with tamoxifen has been demonstrated to be about 50-fold greater in rats or mice compared with humans. This is of special note since alpha-hydroxytamoxifen appears to be the major metabolite leading to the formation of DNA adducts in rats.[14,15,25] In addition, the 3',4'-epoxy metabolite and its hydrolyzed metabolite 3',4'-dihydrodihydroxytamoxifen have been identified in rat liver microsomal systems following administration of tamoxifen, but have not been not identified in microsomal systems from mice or humans. Only trace amounts of the 3,4-epoxy metabolite and its hydrolyzed derivative 3,4-dihydrodihydroxytamoxifen have been identified in a human liver microsomal system.
In Vitro Studies
Human and rat liver microsomes have the enzymatic capability of activating tamoxifen to electrophilic metabolites that can form DNA adducts. However, the quantitative relationships between the number of DNA adducts formed per unit dose of tamoxifen are several orders of magnitude lower in human cells than in cells of rats. [19,25,28-30] Large differences exist between the number of tamoxifen-associated DNA adducts produced in vitro using rat hepatocytes compared to the number formed using human hepatocytes. In a recent study, DNA adducts on the order of 18 to 90 adducts per 108 nucleotides were formed when rat hepatocytes were treated with 1-10 mM tamoxifen. In contrast, no adducts were detected in similarly treated human hepatocytes (assay detection limit of one adduct per 1010 nucleotides). Similarly, human hepatocytes treated with alpha-hydroxytamoxifen produced 300-fold fewer DNA adducts than did rat hepatocytes. Very low numbers of DNA adducts (one adduct per 109 nucleotides) were formed by tamoxifen or toremifene(Drug information on toremifene) (another antiestrogen) when human or rat liver microsomes were incubated with salmon sperm DNA and 1% solutions of the antiestrogen compound, or when human lymphocytes were incubated with 0.1% or 1% solutions of the antiestrogens. When tamoxifen at relatively high concentrations (20 to 500 mM) was incubated with endometrial tissue from nonexposed women, no DNA adducts were detected using a method to detect tamoxifen-specific DNA adducts. DNA adduct levels on the order of 5 to 30 adducts per 108 nucleotides were observed following treatment of endometrial tissue with alpha-hydroxytamoxifen (20 to 500 mM); however, the lowest dose of alpha-hydroxytamoxifen required to produce DNA adducts represented a concentration 10,000 times higher than that observed in the endometrial cultures of tamoxifen-treated patients, or found circulating in the blood of patients. Incubation of 100 mM 4-hydroxytamoxifen or metabolite E with rat uterine peroxidase or horseradish peroxidase produced 4 to 55 DNA adducts per 109 nucleotides, with higher numbers of adducts being found in extracts from tamoxifen-treated rats.