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Clinical Relevance of Laboratory and Animal Data on Tamoxifen

Clinical Relevance of Laboratory and Animal Data on Tamoxifen

ABSTRACT: Tamoxifen is being evaluated in clinical trials as a preventive agent in women at high risk for breast cancer. This new, potentially long-term therapeutic role has generated some concerns regarding safety, based on the results of animal studies that demonstrated an increased incidence of liver cancer in rats and the formation of DNA adducts. However, direct extrapolation of data from rats to risk estimates in women does not appear to be appropriate. Studies have shown that there are substantial species-related differences in activation and detoxication of tamoxifen that directly affect the formation of DNA adducts. This major species difference in DNA adduct formation appears to be the result of differences in metabolic activation and detoxication; humans form much less of the electrophilic metabolites that bind to DNA and rapidly detoxify those that are formed. [ONCOLOGY 11(Suppl 1):39-44, 1997]

Introduction

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 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 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.[2] 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.[2]

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.[10] In contrast to the rat data, epidemiologic
observations in humans do not suggest an increased risk for liver tumors
in patients treated with tamoxifen.[21] 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.

Multistage Concept
of Carcinogenesis

Carcinogenesis is known to be a multistage process involving multiple
genetic alterations in key genes involved in cell regulation and growth.
[22] 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.[12] 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]

Formation
of DNA Adducts Associated With Tamoxifen

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.[15]

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.[25] 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.[27] 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.[27]

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.[25] 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.[25] Very
low numbers of DNA adducts (one adduct per 109 nucleotides)
were formed by tamoxifen or 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.[31] 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.[30] 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.[30] 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.[32]

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