INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
TOXICOLOGICAL EVALUATION OF CERTAIN
VETERINARY DRUG RESIDUES IN FOOD
WHO FOOD ADDITIVES SERIES: 43
Prepared by the Fifty-second meeting of the Joint FAO/WHO
Expert Committee on Food Additives (JECFA)
World Health Organization, Geneva, 2000
IPCS - International Programme on Chemical Safety
PRODUCTION AIDS
ESTRADIOL-17ß, PROGESTERONE, AND TESTOSTERONE
First draft prepared by
J. Leighton, S. Franceschi, G. Boorman, D.W. Gaylor, and J.G. McLean
Estradiol-17ß
Explanation
Biological data
Absorption, distribution, and elimination
Biotransformation
Hydroxylation
Conjugation
Biochemical parameters
Synthesis
Mechanism of action
Toxicological studies
Acute toxicity
Short-term studies of toxicity
Long-term studies of toxicity and carcinogenicity
Genotoxicity
Reproductive toxicity
Special studies on mechanism of action
Observations in humans
Therapeutic use
Estradiol-related genetic markers of carcinogenicity
Progesterone
Explanation
Biological data
Absorption, distribution, and excretion
Biotransformation
Biochemical parameters
Synthesis
Mechamism of action
Toxicological studies
Acute toxicity
Short-term studies of toxicity
Long-term studies of toxicity and carcinogencity
Genotoxicity
Reproductive toxicity
Observations in humans
Testosterone
Explanation
Biological data
Absorption, distribution, and elimination
Biotransformation
Biochemical parameters
Synthesis
Mechamism of action
Toxicological studies
Acute toxicity
Short-term studies of toxicity
Long-term studies of toxicity and carcinogenicity
Genotoxicity
Reproductive toxicity
Observations in humans
Epidemiological studies of women exposed to postmenopausal estrogen
therapy and hormonal contraceptives
Methods
Postmenopausal oestrogen therapy
Exposure
Human carcinogenicity
Breast cancer
Endometrial cancer
Cervical cancer
Ovarian cancer
Cancers of the liver and biliary tract
Colorectal cancer
Cutaneous malignant melanoma
Thyroid cancer
Summary and conclusions
Cardiovascular disease
Osteoporosis
Overall mortality
Hormonal contraceptives
Exposure
Human carcinogenicity
Breast cancer
Endometrial cancer
Cervical cancer
Ovarian cancer
Cancers of the liver and biliary tract
Colorectal cancer
Cutaneous malignant melanoma
Thyroid cancer
Summary and conclusions
Cardiovascular disease
Acute myocardial infarct
Stroke
Venous thromboembolism
Overall mortality
Meat intake and cancer risk
Comments and evaluation
Estradiol-17ß
Progesterone
Testosterone
References
The purpose of this monograph is to provide a review and summary
of the scientific information relative to a toxicological assessment
of the safety of three endogenous hormones, estradiol-17ß,
progesterone, and testosterone, with emphasis on information published
since the review of the Committee at its thirty-second meeting (Annex
1, reference 80). The biology and toxicology of the compounds and
metabolites formed endogenously and ingested orally are summarized. As
the pharmacokinetics and pharmaco-dynamics of synthetic steroidal and
nonsteroidal substances (e.g. diethyl-stilbestrol) differ
substantially, only a limited discussion of the pharmacology of these
compounds is presented. This review is not intended to be exhaustive
but to highlight the scientific literature that may be relevant to use
of the hormones from the point of view of food safety. The Committee
at its thirty-second meeting did not prepare toxicological monographs
on the natural hormones estradiol-17ß, progesterone, and testosterone.
1. ESTRADIOL-17b
1.1 Explanation
Estradiol benzoate (10-28 mg) or estradiol-17ß (estradiol; 8-24
mg) are administered to cattle as an ear-implant formulation to
increase the rate of weight gain (i.e. growth promotion) and to
improve feed efficiency. Estradiol valerate is administered by
subcutaneous or intramuscular injection to synchronize estrus in
cattle. Esters of estradiol are rapidly cleaved to estradiol in vivo
and are thus also considered to be endogenous substances, as the
residues produced are structurally identical to the estradiol produced
in animals and humans after hydrolysis.
Estradiol was reviewed previously by the Committee, at its
thirty-second meeting (Annex 1, reference 80), when it concluded that
the establishment of an acceptable residue level and an ADI was
'unnecessary'. This conclusion was based on studies of the patterns of
use of estradiol for growth promotion in cattle, the residues in
animals, analytical methods, toxicological data from studies in
laboratory animals, and clinical findings in human subjects. The
Committee further concluded that estradiol residues resulting from its
use for growth promotion in accordance with good husbandry practices
were unlikely to be a hazard to humans.
1.2 Biological data
1.2.1 Absorption, distribution, and excretion
Estradiol is generally considered to be inactive when
administered orally due to gastrointestinal and/or hepatic
inactivation. In a study to monitor its oral availability and to
identify the sites of metabolism, 14C-estradiol was infused into
selected portions of the gastrointestinal tract of gilts, and blood
samples were collected from the jugular and portal veins. The
concentration of free estrogens in the jugular vein was low (< 1%) at
all times after instillation of labelled estradiol, and it was
detectable only briefly. The concentration of conjugated estrogens in
the jugular vein peaked rapidly after instillation, particularly when
instilled into the lower gut. Approximately 60-90% of the radiolabel
in blood was present as glucuronide conjugates; smaller amounts of
sulfated compounds were detected, and approximately 1% as
diconjugates. The principal steroid identified after cleavage by
b-glucuronidase and sulfatase was estrone. The authors concluded that
conjugation occurs as estradiol crosses the mucosa of the
gastrointestinal tract, and free estradiol in the portal plasma is
conjugated during the first pass through the liver (Moore et al.,
1982). In companion studies, the authors concluded that the limiting
factor in absorption of conjugates was hydrolysis to free estrogen
(Pohland et al., 1982) and that a possible dose-limiting rate of
absorption was observed at the highest dose (4 mmol 3H-estradiol
glucuronide) (Coppoc et al., 1982).
Crystalline estradiol (10 mg in cocoa butter) was placed in the
stomachs of prepubertal gilts that had been held without food for 26
h, and blood samples were taken from the jugular and hepatic portal
veins for hormone measurements. The concentrations of estradiol,
estrone, estradiol glucuronide, and estrone sulfate in the hepatic
portal vein rose within 5 min and remained elevated for several hours.
Estradiol represented only 6% of the total estrogen measured during
the sampling period, indicating extensive pre-hepatic metabolism of
estradiol. In the periphery, the concentrations of estradiol
glucuronide, estrone glucuronide, and estrone sulfate, but not those
of estradiol or estrone, rose in the jugular vein, indicating that
most of the estradiol and estrone had been removed by the liver.
Infusion of bile containing estrogens into the duodenum resulted in
peaks of estrogen glucuronide and estrone glucuronide in the hepatic
portal and jugular veins within a few minutes, followed by a second
rise 180 min later. The first peak did not occur in bile extracted
with ether to remove free estradiol and estrone, and the second peak
did not occur in gilts given oral antibiotics before bile infusion.
The authors concluded that estrogens administered orally are
conjugated by the gut wall and pass to the liver, where they enter
either the bile pool for enterohepatic circulation or the bloodstream
(Ruoff & Dziuk, 1994).
Oral administration of 0.5 mg fine-particle estradiol in the
early follicular phase of the menstrual cycle to six fasting, female
volunteers resulted in a peak mean estradiol concentration of 211
pg/ml 4 h after administration (mean basal estradiol concentration,
138 pg/ml). The serum estrone concentrations also peaked at this time,
when the peak:baseline ratio of estrone was greater than that of
estradiol. Peaks were observed 4 h after dosing for estrone sulfate
and 6 h after dosing for estradiol sulfate; the peak for estrone
sulfate was always higher than that for estradiol sulfate. The
predominance of estrone over estradiol in serum after oral
administration of estradiol and comparison with serum concentrations
reached after vaginal administration indicate extensive first-pass,
probably intestinal, metabolism (Nahoul et al., 1993).
The distribution of estradiol in female Wistar rats was measured
in heart, liver, kidney, brain, and plasma by radioimmunoassay for 24
h after intravenous administration of 0.1 mg/kg bw or after
intragastric administration of 10 mg/kg bw. The concentration of
estradiol in liver was 20 times higher after intragastric than after
intravenous administration when equivalent plasma concentrations of
hormone were evaluated. Negligible differences were seen in the
estradiol concentrations of other tissues. The tissue concentrations
of estradiol were higher than those in plasma at all times. The
absolute bioavailability, as measured by comparison of the
dose-corrected values for the area under the integrated
concentration-time curve (AUC), was 8.3% after an intragastric dose of
10 mg/kg bw. The total clearance was 154 ml/min per kg bw. The
half-life of estradiol in liver was 2.6 h (Schleicher et al., 1998).
The uptake of estradiol by adipose tissue, a reservoir for estrogens,
was not investigated in this study.
Fourteen young women received a single dose of 2, 4, or 8 mg
estradiol orally or 0.3 mg intravenously. The 8-mg dose of estradiol
resulted in a 70-78% reduction in the AUC relative to expected
values for estradiol and for free and total estrone, suggesting
incomplete absorption at this dose. The absolute bioavailability of
the 4-mg dose was calculated to be 5%. The mean ratio of free
estrone:estradiol was 1 after intravenous injection and and 20 after
oral administration. In a two-comparment model, the AUC for young
women given a 0.3-mg dose intravenously was 4000 pg-h/ml; total
clearance was 22 ml/min per kg bw. Pharmacokinetic parameters showed
high intraindividual and interindividual variation,which limits the
therapeutic usefulness of oral preparations (Kuhnz et al., 1993).
Circulating estradiol is bound to sex hormone-binding globulin
(SHBG) and, to a lesser extent, serum albumin. Only 1-2% of
circulating estradiol is unbound; 40% is bound to SHBG and the
remainder to albumin (Carr, 1998). Plasma SHBG is secreted from the
liver; a similar, non-secretory form is present in many tissues,
including reproductive tissues and the brain. Adult rodent livers do
not produce the secretory form of SHBG (Reventos et al., 1993). Some
estrogen metabolites (2-methoxyestrone and 2-methoxy-estradiol) have
higher binding affinities for SHBG than estradiol itself (Philip &
Murphy, 1986), and other estrogens (estrone and estriol) do not bind
to this serum protein in humans (Renoir et al., 1980). Estradiol binds
to human SHBG with lower affinity than testosterone.
The plasma concentrations of SHBG are regulated; they may be
increased 5-10-fold by estrogens and decreased twofold by testosterone
(Griffin & Wilson, 1998). Thus, a 20-fold higher concentration of
total testosterone in men than in women results in a 40-fold
difference in free testosterone (Grumbach & Styne, 1998). Unliganded
plasma SHBG binds to either steroid or to SHBG-receptor; SHBG must
first bind to the receptor and then the steroid in order to act: SHBG
that is liganded to steroid cannot bind to the receptor (Hyrb et al.,
1990). The SHBG-receptor complex present on the membranes of target
tissues may be responsible for the interaction between the steroid
hormone and cAMP pathways (Rosner, 1991). These observations provide a
mechanistic explanation for the finding that some estrogenic effects
are rapid (milliseconds) and are possibly mediated in a non-genomic
manner. The intracellular form of the SHBG protein may sequester or
direct hormone to the target tissue.
Estrogens are eliminated in faeces and urine. The principal
metabolites found in urine are polyhydroxylated forms conjugated at C3
to glucuronic acid or sulfate. Elimination in bile is subject to
enterohepatic circulation, and 20% of estrogens may be lost through
faecal elimination. A high-fibre diet has been implicated in increased
elimination of estrogens by this route, probably by decreasing gut
transit time (Lewis et al., 1997). A high-fibre diet nonsignificantly
lowered the serum estradiol AUC in human volunteers given an oral dose
of estradiol glucuronide (Lewis et al., 1998).
Urinary and faecal metabolites of estrogens in animals and humans
have been studied for use as possible indicators of risk for
hormone-dependent cancers or for infertility. Quantitative and
qualitative differences between low-and high-risk populations and
alterations in metabolite profiles due to diet have been reported
(Michnovich & Bradlow, 1990; Aldercreutz et al., 1994; Ursin et al.,
1997). There is at present no consensus about the importance of
specific metabolites or metabolite ratios as prognostic factors, with
the possible exception of estriol as a marker of the well-being of the
feto-placental unit.
The terminal plasma half-life of estradiol after intravenous
adminis-tration to humans was 27 min; the volume of distribution was
calculated to be 0.082 l/kg bw (White et al., 1998). Elsewhere, the
plasma half-life of estradiol has been reported to be approximately 30
min (Wingard et al., 1991).
1.2.2 Biotransformation
The major metabolites of estradiol, progesterone, and
testosterone are shown in Figure 1.
1.2.2.1 Hydroxylation
Concern about the carcinogenicity of estrogens and, more
recently, the possible genotoxicity of estrogen metabolites has
sparked interest in establishing the pathways of estradiol metabolism,
and extensive reviews have been published (IARC, 1979; Zhu & Conney,
1998a). The two main competing, irreversible pathways for estradiol
hydroxylation are 2-or 4-hydroxylation and 16 alpha-hydroxylation
(Michnovicz et al., 1989), which have been implicated in both the
pathophysiology and the protective characteristics of estrogens. Minor
pathways of hydroxylation at other sites in the steroid metabolic
pathway have also been identified (Zhu & Conney, 1998a).
Hepatic hydroxylation of estradiol in humans and most other
species leads primarily to the formation of 2-hydroxyestradiol or
2-hydroxyestrone, with subsequent methylation; 4-hydroxy estrogens are
also formed, although to a lesser extent. In the alternative pathway,
the principal products are 16 alpha-hydroxyestrone and estriol, both of
which are estrogen agonists. The pathway of estradiol metabolism in
vitro was shown to be concentration-dependent; in hamster liver
microsomes, 16alpha-hydroxylation predominates at low (< 25 µmol/L)
concentrations, whereas 16 alpha-and C2-hydroxylation contributed
equally to estradiol metabolism at higher concentrations (Butterworth
et al., 1996). Human forms of cytochrome P450s (CYP) which catalyse
the 2-or 4-hydroxylation of estradiol and estrone include CYP1A2 and,
to a lesser extent, CYP3A4 and CYP2C9 (Shou et al., 1997; Yamazaki et
al., 1998). Estradiol and estrone 16a-hydroxylation is catalysed by
CYP1A2 (estradiol) and CYP3A4 (estradiol and estrone). CYP1B1
catalyses the 4-hydroxylation of estrone and estradiol and may be the
dominant enzymatic pathway for estrogen metabolism in some
extrahepatic tissues, particularly steroidogenic tissues and their
respective targets (Larsen et al., 1998; Zhu & Conney, 1998a).
While most estrogen metabolism occurs in the liver as
2-hydroxylation, extrahepatic metabolism occurs as well. Conflicting
reports have been published on the predominance of 2-and
4-hydroxylation of estradiol in Syrian hamster kidney. The major route
appears to be 2-hydroxy formation after catalysis by CYP1A1/2 and
CYP3A expressed in this tissue (Hammond et al., 1997; Sarabia et al.,
1997). Alternatively, 4-hydroxylation has been shown to predominate
over 2-hydroxylation in the hamster kidney (Weisz et al., 1992). The
4-hydroxy estradiol formed in this tissue is thought to be due to the
lack of specificity of the responsible CYPs, as a specific estrogen
4-hydroxylase (presumably CYP1B1) was not found in this tissue. CYP1B1
protein was also not found in human renal adenocarcinoma cells (Spink
et al., 1997). Rat pituitary, mouse, and human uterus and human
mammary gland are other tissues that express high levels of estrogen
4-hydroxylase (Liehr et al., 1995; Liehr & Ricci, 1996; Yager & Liehr,
1996; Larsen et al., 1998).
Significant differences in steroid metabolism are seen between
rodents and humans (IARC, 1979). Sex-specific regulation of CYPs has
been observed in rodent but not human liver, although sex differences
in the metabolism of xenobiotics are found in humans (Kedderis &
Mugford, 1998). Human but not mouse CYP1B1, recently identified as an
estrogen 4-hydroxylase, metabolizes estradiol (Savas et al., 1997).
Several mechanisms of CYP-mediated aromatic hydroxylation of
estrogens (estradiol and estrone) have been proposed, including
epoxide formation, direct oxygen insertion, and hydrogen abstraction.
Hydroxylation by hydrogen abstraction, electron delocalization, and
subsequent hydroxy radical addition has been proposed on the basis of
electronic considerations of oxidation of estrone and substrates with
additional aromaticity (2-napthol and equilenin) (Sarabia et al.,
1997).
Hydroxyestrogens may be further modified by the action of
catechol-O-methyltransferase (COMT). High activity of COMT is found in
many tissues, including liver and kidneys, blood cells, endometrium,
and breast. A genetic polymorphism for this enzyme results in a
trimodal distribution of activity, but epidemological studies of the
polymorphism in relation to breast cancer risk have yielded
conflicting results (Lavigne et al., 1997; Millikan et al., 1998;
Thompson et al., 1998). The methylation of catecholestrogens
effectively prevents these compunds from entering the redox cycling
pathway, and 2-methoxyestradiol may be an antitumour agent (Zhu &
Conney, 1998a,b).
Methylation of 4-hydroxyestradiol by COMT is inhibited by
2-hydroxy-estradiol (Roy et al., 1990). Interestingly, tissues which
develop estradiol-induced tumours (rat pituitary, male Syrian hamster
kidney and mouse uterus) have very high concentrations of endogenous
catecholamines (up to 50-fold relative to other strains or species and
non-target tissues). Catecholamines in target tissues may inhibit or
compete for COMT-catalysed methylation, thus leading to increased
concentrations of hydroxylated metabolites of estradiol (Zhu and
Conney, 1998a).
1.2.2.2 Conjugation
Studies of the conjugation of estrogens with glucuronic acid or
sulfate have been reviewed in detail (IARC, 1979). Lysosomes from male
Syrian hamster livers and kidneys can catalyse the deconjugation of
estradiol and estrone glucuronides. The rates of deconjugation of
estrogen glucuronides were higher in kidney than in liver, by 56% for
estrone and 34% for estradiol. Treatment of hamsters for nine days
with subcutaneous implants containing 25 mg estradiol (releasing 61
µg/day) increased lysosomal estrone and estradiol 3ß-glucuronidase
activity in kidney by 15 and 25%, respectively, and by about 100% in
liver. Estradiol was deconjugated at negligible rates in both liver
and kidney (Zhu et al., 1996). Human liver microsomal sulfatases
convert estrone sulfate to estrone before 16 alpha-hydroxylation
(Huang et al., 1998). Estrone sulfate, the most abundant estrogen in
blood, and other estrogen conjugates may serve as a circulating
reservoir of estradiol, and regulation of deconjugation reactions may
affect intracellular estradiol concentrations.
Demethylation of catechol estrogens has also been reported. The
rates of demethylation of 2-and 4-methoxyestradiol were about equal in
kidney microsomes, but the rate of 2-methoxyestradiol demethylation in
liver was fivefold higher than that of 4-methoxyestradiol. Estradiol
treatment decreased hepatic 2-methoxyestradiol demethylation by about
20% relative to controls with little effect on 4-methoxyestradiol
demethylation, whereas the opposite was observed in kidney (Zhu
et al., 1996).
In the absence of conjugation, a pathway for further catalysis of
catechol estrogens has been suggested. Redox cycling of catechol
(hydroxyquinone) to quinone through semiquinone intermediates is
catalysed by oxidation of catechol estrogens by peroxidases or CYP1A1
lipid hydroperoxide cofactors. Reduction of the quinone to the
hydroquinone is catalysed by NADPH-dependent P450 reductase and other
enzymes. Oxygen radicals formed in this redox process may increase the
carbonyl content of proteins, formation of DNA 8-hydroxydeoxyguanosine
adducts, and lipid peroxidation. The authors concluded that redox
cycling is a critical step in estrogen-mediated carcinogenesis (Wang &
Liehr, 1994; Yager & Liehr, 1996).
1.2.3 Biochemical parameters
1.2.3.1 Synthesis
In mammals, estradiol, estrone, and estriol are synthesized from
steroid precursors in the gonads, adrenal cortex, and placenta or
through peripheral conversion of androgens in other tissues of
mammals. Cholesterol, obtained primarily from circulating low-density
lipoprotein, serves as the precursor for steroid biosynthesis,
although steroidogenic cells are capable of local cholesterol
synthesis de novo. In non-pregnant premenopausal women, the
principal estrogens found in the blood are estradiol and estrone.
Estradiol is synthesized and secreted primarily from ovarian granulosa
cells, whereas most estrone (approximately 40% of total estrogens) is
formed peripherally from estradiol and androstenedione. Estradiol and
estrone may be intra converted through the action of the enzyme
17ß-hydroxysteroid dehydrogenase. Estrone may be further metabolized
to estriol, primarily in the liver. Estriol is also formed in the
fetal liver and in the placenta from
16alpha-hydroxydehydroepiandrosterone sulfate, which is secreted from
the fetal adrenal and circulating dehydroepiandrosterone sulfate. At
least 90% of urinary estriol is derived from fetal sources (Carr,
1992). In men and postmenopausal women, the source of serum estradiol
is peripheral conversion of androgens by the enzyme aromatase. In men,
approximately 0.3% of plasma testosterone is aromatized to estradiol;
an additional contribution of approximately 25% of the total estradiol
may be due to testicular secretion (Griffin & Wilson, 1998). Estrone
is the predominant circulating estrogen in postmenopausal women,
formed by peripheral conversion of adrenal androgens in adipose
tissue.
Gonadal synthesis of estradiol is regulated by luteinizing and
follicle stimulating hormones secreted by the anterior pituitary
gland. The secretion of these two hormones is regulated by
gonadotropin-releasing hormone secreted by the hypothalamus, steroid
hormones, and other factors in a complex feedback loop which
effectively regulates the serum concentrations of hormone within a
physiological concentration range, which is particularly variable in
premenopausal women. The feedback loop is controlled by the dominant
circulating hormone (estradiol and progesterone in women, testosterone
in men). Feedback control for estradiol in men and for testosterone in
women is therefore not operative (Wilson et al., 1998). There is
evidence that this feedback loop exists in prepubertal children but is
quiescent.
In humans, plasma estradiol concentrations generally remain low
during the first 12 years of life. Around the time of menarche, rising
plasma concentrations of gonadotropins from the anterior pituitary
stimulate the ovary to produce estradiol. During a normal menstrual
cycle, plasma estradiol concentrations change very little throughout
the first half of the follicular phase but increase as the follicles
develop, reaching serum concentrations that are up to nine times
greater than the basal concentrations near mid-cycle. After the
mid-cycle surge, the estradiol concentrations fall precipitously.
During the luteal phase, the serum estradiol concentrations rise to a
plateau for 8-10 days, before declining. Should fertilization occur,
the corpus luteum formed from the dominant follicle after ovulation
remains active as the principal source of estradiol for the first 6-8
weeks of pregnancy. The corpus luteum is later supplanted by the
placenta as the site of estrogen synthesis. As the placenta lacks
17 alpha-hydroxylase, fetal and maternal circulating androgens are
necessary for placental estrogen synthesis. During pregnancy, the
feto-placental unit secretes a large quantity of estriol into the
maternal circulation, which is ultimately excreted in the urine.
The concentrations of circulating estrogens, their daily
production, and their metabolic clearance rates can be found in
previous reviews (IARC, 1979, specifically 'General remarks on sex
hormones') and in most textbooks of endocrinology (e.g. Braunstein,
1994; Goldfien & Monroe, 1994; Carr, 1998; Griffin & Wilson, 1998; see
also textbook appendices for summary tables). They are summarized in
Table 1. Somewhat different values can be calculated for the basal
production rate of estradiol in prepubertal boys and girls
(Angsusingha et al., 1974; IARC, 1979) when measurements of hormone in
urine or plasma are used as the basis for the calculation (4 or 12
mg/day).
1.2.3.2 Mechanism of action
The conventional view of the action of steroid sex hormones
involves interaction of the sex hormone with specific intracellular
receptors, which subsequently bind tightly to specific DNA sequences
in the genome (Malayer & Gorski, 1993). This tight nuclear binding
initiates transcription of specific genes, which ultimately leads to
physiological events. These include include development of
reproductive tissues, maturation of the ovarian follicle, development
of the uterus and vagina, and ductal development in the breast.
Estrogen withdrawal results in menstruation. In non-reproductive
tissues, estrogens may affect bone growth and prevent bone resorption,
and effects on the plasma lipid profile through action in the liver.
Estrogens typically promote cell growth or cell proliferation in
responsive cells in culture.
Table 1. Levels of circulating estrogens, metabolic clearance rates, and
daily production
Sex Age or phase Serum Metabolic Total daily
concentration clearance production
(pg/ml) (L/day) (mg/day)
Male 1400
Prepubertal < 10 < 0.014
12-16 years < 23 < 0.031
> 16 years 20-50 0.027-0.068
Female 1400
< 8 years < 7 < 0.01
2-12 8-18 0.01-0.024
12-14 16-34 0.02-0.09
14-16 20-68 0.03-0.09
Early follicular 20-100 0.03-0.14
Preovulatory 100-350 0.14-0.47
Luteal 100-350 0.14-0.47
Late pregnancy 18 000 24
Postmenopausal 10-30 0.01-0.04
It has been proposed that the carcinogenicity of estrogens is
distinct from its hormonal properties; however, since that hypothesis
originated, information on estrogen-receptor and ligand interactions
has been re-evualuated in the light of recent identification of
additonal estrogen receptor proteins. Three specific receptors have
now been identified for the endogenous ligand estradiol: the classical
receptor ER alpha, ERß, and ERß2. Analyses of ER alpha and ERß RNA
indicate that ER alpha is widely distributed and that ER ß is
prominent in prostate, ovary (localized to the granulosa cells of the
maturing ovary), epididymis, urinary bladder, uterus, lung, thymus,
colon, small intestine, vessel walls, pituitary glan, hypothalamus,
cerebellum, and brain cortex (Couse et al., 1997a; Kuiper et al.,
1998). These receptor isoforms differ in their agonist and antagonist
reactions to agents such as tamoxifen and to classes of agents
variously referred to as 'endocrine disruptors' or 'xenoestrogens' and
to endogenous estrogen metabolites such as 2-hydroxyestradiol.
Moreover, alpha-estrogen receptor knockout (alpha ERKO; Couse et al.,
1997a) and ERß-/- female mice (Krege et al., 1998) have very different
phenotypes. Alpha ERKO females lack the ability to complete
folliculogenesis, are infertile, and have multiple cystic,
haemorrhagic, and atretic follicles, whereas ERß-/- female mice
develop normally and are indistinguishable from their wild-type
littermates. These mice are fertile, but their fertility is
compromised, as demonstrated by reductions in litter size. Mammary
gland development is normal in ERß-/- mice, in contrast to the absence
of breast development beyond that of prepubertal females in alpha ERKO
mice. Male mice lacking ERß are also fertile (alpha ERKO males are
infertile) but develop prostate and bladder hyperplasia as they age.
These ERKO mice and the receptor-binding properties of various ligands
demonstrate that ER alpha and ERß have different functional
responsibilities.
Abundant evidence exists that hormonal carcinogenicity is linked
to the relative balance of various estrogens. Proliferation of mammary
glands and other reproductive (i.e. target) tissues is inextricable
linked to hormonal changes during the menstrual cycle, pregnancy, and
the initiation or cessation of cyclic menstruation. The cyclic
proliferation of tissues is correlated to the cellular content of the
estrogen receptor. In cultured cells, the growth of cells with
estrogen receptors (e.g. MCF-7 cells) but not those without estrogen
receptors (e.g. MDA-MB-231 cells) is dependent on the estradiol
concentration in serum. Molecular genetic studies of human cancer
indicate that the progression from a normal to malignant phenotype
requires activation of one or several oncogenes and/or inactivation of
tumour suppressor genes. These events require cell division (reviewed
by Bernstein & Ross, 1993; Russo & Russo, 1996; Tsai et al., 1998).
A functional role for catechol estrogens distinct from that for
estradiol has been suggested (reviewed by Zhu & Conney, 1998a,b).
Because of the extremely short half-lives of these compounds
(Schneider et al., 1984), they are unlikely candidates for circulating
hormones. Locally, 2-hydroxyestradiol reportedly stimulates
progesterone production in ovarian granulosa cells (Spicer et al.,
1990), and 2-hydroxyestrone has been shown to inhibit MCF-7 cell
proliferation in the presence of quinalizarin, a potent inhibitor of
COMT. This challenge can be overcome with physiological concentrations
of estradiol. No effects on cell growth were observed with
concentrations of 10-9 to 10-6 mol/L of 2-hydroxyestradiol in the
absence of methyltransferase inhibition (Schneider et al., 1984).
The endogenous metabolite 2-methoxyestradiol has been suggested
to be an antiangiogenic factor and tumour suppressor (Fotis et al.,
1994; Zhu & Conney, 1998a); however, the concentrations required to
induce apoptotis in cultured cells are about 10 times greater than
those observed in humans (Yue et al., 1997). Those authors suggested
that unless the concentration of 2-methoxyestradiol in the lipid phase
(i.e. membranes) exceeds that in the aqueous phase, as reported for
some lipophilic calcium blockers, the antiangiogenic properties of
2-methoxyestradiol are of little physiological relevance.
A role for catechol estrogens in implantation in the mouse uterus
has been suggested on the basis of the observation of increased
activity of 4-hydroxylase activity on day 4 of pregnancy (Paria et
al., 1990). 2-and 4-Hydroxyestradiol bind to the estrogen receptor
with reduced relative affinities of 23 and 26%, respectively (Schultze
et al., 1994). Additionally, a distinct signaling pathway separate
from the estrogen receptor may exist for 4-hydroxyestradiol. In
alpha ERKO mice, 4-hydroxyestradiol stimulated up-regulation of
lactoferrin mRNA and water imbibition (Das et al., 1997). In these
mice, the effects of 4-hydroxyestradiol could not be mimicked by
estradiol, nor could the effects be blocked by the anti-estrogen ICI
182,780; however, it has been postulated that the estrogenic effects
in the uterus in alpha ERKO mice are mediated through ERß (Krege et
al., 1998).
The physiological effects of estradiol that are reported not to
be receptor-mediated (i.e. not mediated via the classical receptor
mechanism) include those on myometrial, neuronal, pituitary, maturing
oocyte, and granulosa cells (Wehling, 1997). Estradiol may protect
against oxidative damage in neuronal cells (Behl et al., 1995), but
very high concentrations of estradiol were required for this effect:
10-5 but not 10-7 mol/L was effective in preventing cell death. At
physiological concentrations in blood, the antioxidant properties of
estradiol protect against oxidation of low-density lipoprotein (Rifici
& Kachadurian, 1992; Hoogerbrugge et al., 1998). Novel so-called
'scavestrogens', structurally related to 17 alpha-estradiol, have
antioxidant properties that protect against the radical-mediated death
of cultured cells (Blum-Degen et al., 1998). Other studies indicate
that the pro-oxidant and antioxidant properties of estrogens may be
dependent on structure and concentration. Estradiol, estriol, and
methoxyestrogen metabolites had only antioxidant properties. Catechol
estrogens showed pro-oxidant properties at low concentrations (5
pmol/L to 100 nmol/L), but antioxidant properties dominated at high
concentrations (0.5-50 µmol/L) (Markides et al., 1998). In addition to
oxidant and antioxidant effects, a rapid, non-genomic effect of
estradiol, possibly mediated by cAMP, Ca++, or ion channel gating,
has been postulated (reviewed by Moss et al., 1997).
1.3 Toxicological studies
1.3.1 Acute toxicity
The therapeutic dose of fine-particle estrogen given orally is
0.5-2 mg/day. No adverse effects were reported in children after
accidental ingestion of large doses of estrogen-containing oral
contraceptives (Physician's Desk Reference, 1999). An
electroencephalogram of a young woman who took 160 mg of estradiol
valerate (80 tablets of 2 mg), which is converted to estradiol-ß
in vivo, showed traces typical of subcortical disturbance on the
first day; however, the recording was normal one week later (Punnenon
& Salmi, 1983).
1.3.2 Short-term studies of toxicity
Estradiol was administered in the diet to female Crl:CD BR rats
at doses equal to 0. 0.003, 0.17, 0.69, or 4.1 mg/kg bw per day for 90
days. The end-points were chosen to evaluate both short-term and
reproductive toxicity and several mechanistic and biochemical
parameters. Administration of doses > 0.17 produced dose-dependent
increases in body weight, food consumption, and feed efficiency. At
0.69 and 4.1 mg/kg bw per day, minimal to mild non-regenerative
anaemia, lymphopenia, decreased serum cholesterol (at the high dose
only), and altered splenic lymphocyte subtypes were observed. Changes
in the weights of several organs were noted. Evidence of ovarian
malfunction (reduced corpora lutea and large antral folicles) was
found at doses > 0.17 mg/kg bw per day. Pathological changes in
males and females fed 0.69 or 4.1 mg/kg bw per day included
centrilobular hepatocellular hypertrophy, diffuse hyperplasia of the
pituitary gland, feminization of the male mammary gland, mammary gland
hyperplasia in females, cystic ovarian follicles, hypertrophy of the
endometrium and endometrial glands in the uterus, degeneration of the
seminiferous epithelium, and atrophy of the testes and acessory sex
glands (Biegel et al., 1998b).
1.3.3 Long-term studies of toxicity and carcinogenicity
The toxicity of estrogens, including estradiol and related
esters, has been reviewed extensively by working groups convened by
the IARC (1979, 1987). After reviewing studies of estradiol
administered orally to mice and by subcutaneous injection or
implantation in mice, rats, hamsters, guinea-pigs, and monkeys, the
group concluded that there is sufficient evidence for the
carcinogenicity of estradiol in experimental animals, noting that:
"Administration to mice increased the incidences of mammary,
pituitary, uterine, cervical, vaginal, testicular, lymphoid and bone
tumours. In rats, there was an increased incidence of mammary and/or
pituitary tumours. estradiol-17b produced a statistically
nonsignificant increase in the incidence of foci of altered
hepatocytes and hepatic nodules induced by partial hepatectomy and
administration of N-nitrosodiethylamine in rats. In hamsters, a high
incidence of malignant kidney tumours occurred in intact and castrated
males and in ovariectomized females, but not in intact females. In
guinea-pigs, diffuse fibromyomatous uterine and abdominal lesions were
observed.' The IARC working group concluded that the carcinogenic
effects of estrogens and progestogens were inextricably linked to the
hormonal milieu and to dose-effect relationships (IARC, 1987).
Hormonal effects on non-cancer end-points were not evaluated by the
groups.
The induction of renal tumours by various steroidal and
non-steroidal estrogens was examined in castrated male Syrian hamsters
treated subcutaneously for nine months with a capsule that released an
average of 110 µg of the hormone per day. The tumour incidences
associated with the hormonal activity of the substances tested are
presented in Table 2. These data demonstrate a good correlation among
the hormonal parameters progesterone receptor induction and serum
prolactin and relative estrogenic potency (estrogen receptor binding)
in hamster kidney. All animals trested with estrone, equilin plus
d-equilin, or Premarin(R) developed renal tumours, with combined
numbers of tumour foci in both kidneys of 15, 18, and 16, respectively
(Li et al., 1995).
Castrated adult male Syrian hamsters were treated for eight
months with subcutaneous pellets containing 20 mg estrogen or
anti-estrogen; the release rates in µg/day were as follows:
diethylstilbestrol, 156; ethinyl-estradiol, 215; estradiol, 96;
estradiol-17 alpha, 104; 17 alpha-ethinyl-11ß-methoxy-estradiol
(moxestrol), 104; and tamoxifen, 183. Treatment with ethinylestradiol
resulted in progressive dysplasia but no renal tumours, but dysplasia
was observed in the proximal tubules of the renal cortex, which was
uncommon in animals treated with diethylstilbestrol or estradiol.
Animals treated with estradiol, diethylstilbestrol, or moxestrol had a
tumour incidence of 100%, which was completely abolished by
concurrrent treatment with ethinylestradiol (Table 3). Simultaneous
administration of diethylstilbestrol and estradiol-17ß or estradiol-17
alpha (a noncarcinogenic estrogen) did not mitigate the
carcinogenicity of diethylstilbestrol (Li et al., 1998).
The carcinogenicity of estradiol and its metabolites was
investigated in groups of 20-35 B6C3F1 mice of each sex given a
single daily intraperitoneal injection of the compound on four
consecutive days starting at 12 days of age. They were then maintained
for approximately 18 months and killed. Of the catechol estrogens and
their quinones tested, only estrone-3,4-quinone was significantly
carcinogenic in the livers of male mice (Table 4). It was also highly
toxic, as most of the mice died from unknown causes shortly after
treatment. Estrone was protective against liver tumour formation in
this system, and few tumours were induced in female mice (Cavalieri et
al.,1997).
The tumour formation in the kidneys of male Syrian hamsters given
25-mg pellets containing estrogen or catechol estrogen:cholesterol
(90:10) by subcutaneous implantation and killed 175 days later was
studied histologically. Estradiol and 4-hydroxyestradiol each induced
renal tumours in four of five animals, whereas neither
2-hydroxyestradiol nor 2-methoxystradiol was carcinogenic. The lack of
carcinogenicity of 2-hydroxyestradiol was not due to failure of the
hormone to stimulate cell growth in vivo, as estradiol,
4-hydroxyestradiol, and 2-hydroxyestradiol supported the growth of
estrogen-dependent H-301 cells injected into male hamsters.
2-Methoxyestradiol was not effective in stimulating these cells. The
authors note that none of the three compounds was mutagenic in
Salmonella typhimurium TA100 strain (see below; Liehr et al., 1986).
The role of estrogen and its metabolites in tumour formation was
examined in castrated male Syrian hamsters implanted for 9-10 months
with pellets containing various estrogens (doses not given). The
results are shown in Table 5. The authors suggested that the
neoplastic changes seen in hamster kidney after continuous exposure to
estrogens are due to synergistic action between hormonal and
carcinogenic factors (Li & Li, 1989).
Castrated male Syrian hamsters received subcutaneously implanted
pellets containing diethylstilbestrol, alpha-dienestrol, hexestrol,
diethylstilbestrol 3,4-oxide, estradiol, estrone, ethinylestradiol,
equilin or (+)-equilenin, which released 100-210 µg/day, for a total
of nine months. The tumour incidence was 75-100%, except with
ethinylestradiol (20%) and (+)-equilenin (0%). The ability of
estrogens to cause renal tumours correlated well with their ability to
compete for estrogen receptor binding, with the notable exception of
ethinylestradiol (Li et al., 1983). The tumours induced by
ethinylestradiol are of a different origin than other hormone-induced
renal tumours (Oberley et al., 1991). The presence of estrogen
receptors, probably ER alpha, in interstitial cells of control and
estrogen-treated hamsters was confirmed by immuno-histochemical
staining and northern blotting. Receptors were also found in renal
corpuscles, arterial cells, and interstitial capillaries but not in
the tubular epithelia of the cortex, further indicating that the
tumours have an interstitial or mesenchymal origin (Bhat et al.,
1993). The presence of ERß, which was identified after the study, has
not been investigated.
Virgin female C3H/HeJ mice with a high titre of antibodies to the
mouse mammary tumour virus were fed diets that provided doses
equivalent to 0.015, 0.15, or 0.75 mg/kg bw per day estradiol from
week 6 to week 110 of age (Highman et al., 1978). The microscopic
findings in animals sacrificed after 52 weeks of feeding are shown in
Table 6. In a continuation of this study, the authors reported the
preneoplastic and neoplastic findings in mice sacrificed after up to
104 weeks on the estrogenic diets (Highman et al., 1980). The results
are shown in Tables 7 and 8. Uterine cervical adenosis may be a
precursor of cervical adenocarcinoma in C3H/HeJ mice and may therefore
serve as an early indicator of the uterine carcinogenicity of a
compound. In these experiments, high doses of estradiol increased the
incidence of adenosis but did not affect the incidence of ovarian
tubular adenomas. After 66-91 weeks of treatment, high doses of
estradiol also increased the incidence of mammary gland hyperplastic
alveolar nodules but not mammary adenocarcinomas. The authors reported
the occurrence of several other sporadic tumours at different sites in
both control and treated animals. They concluded that the incidence of
lesions in mice given estradiol was generally dose-dependent,
indicating that this compound either induces or facilitates the
development of these lesions (Highman et al., 1980)
Table 2. Estrogenicity and carcinogenicity of various steroidal and stilbene estrogens in Syrian hamster kidney
Estrogen % of competitive Induction of Serum % of animals No. of tumour
binding to estrogen progesterone prolactin with tumours foci in both
receptors due to receptors in kidney (ng/ml) kidneys
renal tumours (fmol/mg protein)
Steroidal
Estradiol-17ß 55 48 390 100 17
11ß-Methoxy ethinyloestradiol 52 60 330 100 22
16 alpha-Hydroxyestrone 48 45 390 38 3
11ß-Methoxyestradiol 30 35 390 25 2
11ß-Methylestradiol 14 18 150 0 0
Estradiol-17 alpha 34 6 130 0 0
Deoxestrone 14 8 94 0 0
Stilbene
Diethylstilbestrol 46 50 450 100 19
Indenestrol B 46 49 280 100 11
Indanestrol 10 29 100 0 0
From Li et al. (1995)
Table 3. Prevention of the carcinogenicity of estrogens by ethinylestradiol
Estrogen % tumour induction No. of tumour
nodules in both
kidneys
Diethylstilbestrol 100 15
Estradiol-17ß 100 13
Ethinylestradiol 10 2
Moxestrol 100 18
Diethylstilbestrol + ethinylestradiol 0 0
17ß-Estradiol + ethinylestradiol 0 0
Moxestrol + ethinylestradiol 0 0
Diethylstilbestrol + diethylstilbestrol 100 12
Diethylstilbestrol + 17ß-estradiol 100 12
Diethylstilbestrol + 17 alpha-estradiol 100 9
From Li et al. (1998)
Intact and ovariectomized or hysterectomized nulliparous female
C3H/HeJ mice, five months of age, received either estradiol at a dose
of 0.5 mg/L in drinking-water for one year or estradiol plus daily
injections of 0.1 mg 2-bromo-alpha-ergocryptine, an effective
suppressor of prolactin secretion. All mice were examined weekly for
mammary tumours. One year after the onset of treatment, all surviving
mice were sacrificed. The formation of mammary hyperplastic nodules
was highly significantly suppressed in mice that received both
estradiol and 2-bromo-alpha-ergocryptine, and the mammary tumour
incidence was slightly but significantly reduced in comparison with
that in animals receiving estradiol alone. The tumour incidence in
concurrent controls was not reported. In a separate study, nulliparous
30-day-old mice received estradiol in the drinking-water with or
without a daily injection of 0.1 mg 2-bromo-alpha-ergocryptine for
19-20 months. The effects on the mammary gland are shown in Table 9.
The authors concluded that at least a portion of the oncogenic
activity of estrogenic steroids on the mammary gland in rodents is
manifested through a stimulatory effect on prolactin secretion
(Welsch, 1976).
The results of short-and long-term studies of the carcinogenicity
of estrogens are summarized in Table 10.
Table 4. Carcinogenicity of estradiol and its metabolites in male
B6C3F1 mice
Compound Dose No. of mice with tumours/
(µmol/kg bw) total no. of animals (%)
Estradiol-17ß 30 6/20 (30)
2-Hydroxy-17ß-estradiol 30 10/28 (36)
4-Hydroxy-17ß-estradiol 30 10/24 (42)
17ß-Estradiol-2,3-quinone 7.5 8/26 (31)
17ß-Estradiol-3,4-quinone 7.5 4/21 (19)
Estrone 30 3/32 (9.4)
2-Hydroxyestrone 30 9/30 (30)
4-Hydroxyestrone 30 8/33 (24)
Estrone-2,3-quinone 7.5 12/25 (48)
Estrone-3,4-quinone 3.7 6/10 (60)
Benzo[a]pyrene 60 12/12 (100)
Solvent 7/19 (37)
Untreated 11/33 (33)
From Cavalieri et al. (1997)
1.3.4 Genotoxicity
A working group convened by IARC evaluated the results of tests
for genetic toxicity conducted with estradiol and concluded:
"Oestradiol-17ß did not induce chromosomal aberrations in bone-marrow
cells of mice treated in vivo. Unusual nucleotides were found in
kidney DNA of treated hamsters. It induced micronuclei but not
aneuploidy, chromosomal aberrations or sister chromatid exchanges in
human cells in vitro. In rodent cells in vitro, it induced
aneuploidy and unscheduled DNA synthesis but was not mutagenic and did
not induce DNA strand breaks or sister chromatid exchanges.
Oestradiol-17ß was not mutagenic to bacteria." The group also
concluded that the limited data available on estrone and estriol were
indicative of genotoxicity (IARC, 1987).
A mechanism has been proposed by which catechol estrogens
interact with DNA. Nonmethylated catechol estrogens can be oxidized to
a quinone which can bind to DNA; thus, 2-and 4-hydroxy estradiol
produce 2,3-and 3,4-quinones, respectively. Reaction of the
3,4-quinone of estrone or estradiol with deoxyguanosine (dG) at N7
resulted in loss of the deoxyribose moiety and thus induced
depurinating adducts. No adducts were observed after reaction of the
3,4-quinone with deoxyadenosine (dA). Reaction of estrone-2,3-quinone
with dG and dA produced a stable N2-dG or N6-dA adduct, the
deoxyribose group remaining intact. Formation of depurinating and
stable adducts in calf thymus DNA by activated catechol estrogens and
in mammary glands of female Sprague-Dawley rats injected with 200 nmol
4-hydroxy-estrone was confirmed by the 32P-postlabelling technique.
The authors suggested that the formation of depurinating adducts via
3,4-quinone followed by misreplication of unrepaired apurinic sites
are the critical steps in initiation of cancer by estrogens (Cavalieri
et al., 1997; Stack et al., 1998).
Studies of the genetic toxicology of estrogens are summarized in
Table 11.
The mutagenicity of estradiol and its metabolites was assessed in
Salmonella typhimurium strain TA100, but the authors concluded that
none of the compounds was mutagenic in this assay (Liehr et al.,
1986). Estradiol was evaluated in several short-term tests for
genotoxic potentiol and at 1, 10, or 100 µg/ml was found to cause
chromosomal aberrations and sister chromatid exchange in cultured
human lymphocytes with and without metabolic activation. In the
absence of metabolic activation, the lowest dose caused aberrations
after 72 h of treatment but not after 24 or 48 h; the intermediate
dose caused aberrations after 48 and 72 h but not after 24 h of
treatment. With a 6-h treatment, aberrations were observed at 10 and
100 µg/ml in the presence of metabolic activation but not in its
absence. Estradiol caused sister chromatid exchange at most doses with
or without metabolic activation (Dhillon & Dhillon, 1995).
Table 5. Carcinogenicity of estradiol and its metabolites in castrated
male Syrian hamsters
Compound No. of mice with tumours/
total no. of animals (%)
Estradiol-17ß 6/6 (100)
Estrone 8/10 (80)
Estriol 4/7 (57)
2-Hydroxy-17ß-estradiol 0/6 (0)
2-Hydroxyestrone 0/6 (0)
4-Hydroxy-17ß-estradiol 5/5 (100)
4-Hydroxyestrone 2/6 (33)
Ethinylestradiol 3/15 (20)
Equilin 6/8 (75)
(+)-Equilenin 0/9 (0)
From Li & Li (1989)
Table 6. Percent incidence of pathological changes in female mice given estradiol for 52 weeks
Dose No. of Effects in the Effects in the Effects in the
(mg/kg bw animals cervix uterine horn mammary gland
per day)
Adenosis in Adenosis in Glandular Hyperplastic Adeno- Osseus
upper third upper and hyperplasia alveolar carcinoma hyperplasia
lower thirds nodules
0 47 11 0 26 0 4 0
0.015 35 17 0 24 0 0 0
0.15 36 15 0 62 3 6 18
0.75 48 38 36 96 9 8 82
From Highman et al. (1977)
Table 7. Incidences (and%) of uterine cervical adenosis and ovarian tubular
adenomas in mice fed estradiol
Dose Week
(mg/kg bw
per day) 26 52 78 104
Adenosis Adenosis Adenosis Tubular Adenosis Tubular
adenoma adenoma
0 2/37 (5) 5/46 (11) 1/14 (7) 2/14 (14) 13/19 (16) 12/24 (50)
0.015 1/37 (3) 5/29 (17) 1/5 (20) 0/5 (0) 3/24 (12) 11/25 (44)
0.15 5/45 (110) 5/35 (14) 3/14 (21) 2/16 (12) 8/20 (40) 12/20 (60)
0.75 25/42 (60) 33/45 (73) 4/7 (57) 0/7 (0) 3/6 (50) 3/7 (43)
Table 8. Incidences (and%) of pathological changes in the mammary gland of female mice fed estradiol
Dose Hyperplastic alveolar nodules Adenocarcinomas
(mg/kg bw
per day) Weeks 0-39 Weeks 40-65 Weeks 66-91 Weeks 92-105 Weeks 0-39 Weeks 40-65 Weeks 66-91 Weeks 92-105
0 0/91 (0) 0/57 (0) 3/29 (10) 6/50 (12) 4/91 (4) 15/57 (26) 13/29 (45) 19/50 (38)
0.015 0/89 (0) 0/63 (0) 0/19 (0) 4/31 (13) 0/89 (0) 28/63 (44) 8/19 (42) 13/31 (42)
0.15 0/94 (0) 2/56 (4) 8/29 (28) 7/21 (33) 4/94 (4) 21/56 (37) 14/24 (58) 6/21 (29)
0.75 0/93 (0) 5/78 (6) 5/19 (26) 6/17 (35) 5/93 (5) 34/78 (44) 11/19 (58) 8/17 (47)
From Highman et al. (1980)
Table 9. Effects of treatment with estradiol with or without 2-bromo-alpha-ergocryptine on the number of mammary hyperplastic
nodules and mammary tumours in young nulliparous C3H/HeJ mice
Treatment No. of mice at No. of mice at No. of hyperplastic nodules No. of mice with
start of study end of study in inguinal mammary glands mammary tumours
Controls 100 16 3.1 11
Estradiol 100 12 4.8 27
Estradiol + 2-bromo-alpha-ergocryptine 100 28 2.8 9
Table 10. Summary of results of short-term and long-term studies
of the carcinogenicity of estrogens
Species Dose Findings Reference
Short-term study
Rats 0.003-4.12 mg/kg bw Histopathological changes, particularly at Biegel et al. (1998b)
per day for 90 days intermediate and high doses
Long-term studies
Mice Increased incidences of mammary, pituitary, IARC (1979)
uterine, vaginal, testicular, lymphoid, and
bone tumours
Rats Mammary and pituitary tumours; statistically IARC (1979)
nonsignificant increase in incidence of foci
of altered hepatocytes and hepatic nodules
after partial hepatotectomy and adminsitration
of N-nitrosodiethylamine
Hamsters Malignant renal tumours in intact and IARC (1979)
castrated males and in ovariectomized
but not intact females
Castrated male 111 µg/day 100% renal tumour incidence Li et al. (1995)
Syrian hamsters,
9 months
Castrated male 96 µg/day 100% incidence of renal tumours; modulated by Li et al. (1998)
Syrian hamsters, ethinylestradiol
8 months
B6C3F1 mice 4 daily Estrone was protective; estradiol-17ß did not Cavalieri et al.
intraperitoneal increase incidence over control (1997)
injections
(30 µmol/kg bw)
Table 10. (continued)
Species Dose Findings Reference
Male Syrian 25 mg subcutaneously, Estradiol-17ß and 4-hydroxy-17ß-estradiol Liehr et al. (1986)
hamsters 175 days produced tumours, but 2-hydroxy-17ß-estradiol
did not
Castrated male Not reported; Estradiol-17ß and 4-hydroxy-17ß-estradiol Li & Li (1989)
Syrian hamsters 9-10 months produced tumours, but 2-hydroxy-17ß-estradiol
did not
Castrated male 100-200 µg/day Ethinylestradiol produced a lower incidence of Li et al. (1983)
Syrian hamsters tumours than estradiol-17ß
Virgin C3H/HeJ 0.015-0.75 mg/kg bw Estradiol-17ß caused a dose-dependent increase Highman et al.
mice per day in feed in tumour incidence (1977, 1980)
C3H/HeJ mice 0.5 mg/l in Estradiol-17ß caused tumours Welsch (1976)
drinking-water
When Swiss albino mice were given a single intraperitoneal
injection of 100, 1000, or 10 000 µg/kg bw, the highest dose increased
the number of micronucleated polychromatic erythrocytes and the
frequency of sister chromatid exchange, although the
polychromatic:normochromatic erythrocyte ratio did not appear to be
affected. Estradiol did not cause reverse mutation in Salmonella
strains TA100, TA1535, TA97a or TA98, at concentrations of 1-10 000
µg/plate in the absence of metabolic activation and 1-1000 µg/plate in
its presence. In a host-mediated assay in which mice were given 100,
1000, or 10 000 µg/kg bw followed 2 h later by injection of
S. typhimurium, no change in the number of His+ revertants per
plate was observed (Dhillon & Dhillon, 1995).
Estradiol was evaluated in five tests for the induction of
micronuclei in bone marrow in vivo in female rats given three daily
subcutaneous doses of 20 µg/kg bw and in mice given a single
intraperitoneal injection of 10-150 mg/kg bw. The authors concluded
that estradiol was not genotoxic to the bone marrow of rodents (Ashby
et al., 1997).
In male B6C3F1 mice and male Fischer 344 rats that received
estradiol at 310, 620, or 1250 mg/kg bw in three injections, there was
no increase in the frequency of polychromatic erythrocytes. In male
and female B6C3F1 mice treated with various numbers of injections,
solvents, routes of administration, and killing schedules, no
significant increase in the frequency of micronuclei was observed
(Shelby et al., 1997).
The onset of genomic rearrangements was tested at 10-5 mol/L
estradiol in two X-ray-transformed cell lines (X-ray-9 and F-17a) and
two untransformed cell lines (10T1/2b and 10T1/2c). Genomic
rearrangements (deletions or additions in minisatellites) were
observed in the transformed but not the untransformed lines. No new
rearrangements were observed after withdrawal of estradiol (Paquette,
1996).
The ability of estradiol to induce morphological transformation,
gene mutations, chromosomal aberrations, sister chromotid exchange,
unscheduled DNA synthesis and other chromosomal changes was assessed
in Syrian hamster embryo cells. Cell growth was completely inhibited
at 10-30 µg/ml but was not affected at a concentration < 3 µg/ml.
Treatment of cells with 0.3-6 µg/ml did not affect their
colony-forming efficiency, but at 10 µg/ml colony formation was 53%
that of controls. Incubation with estradiol at 0.3-10 µg/ml for 48 h
induced a dose-dependent increase in the frequency of morphological
changes. Estradiol in this dose range also induced numerical changes
(chromosome gains and loses). The majority of these cells (94%) were
diploid. Estradiol had no other effects in this assay (Tsutsui et al.,
1987). No exogenous metabolic activation was used in these
experiments.
Table 11. Genetic toxicity of estrogens
End-point Test system Dose Result Reference
In vitro
Reverse mutation S. typhimurium TA100 50-1500 µg/plate Negative Liehr et al.
(1986)
Reverse mutation S. typhimurium TA100, 1-10 000 µg/plate -S9 Negative Dhillon & Dhillon
TA1535, TA97a, TA98 1-1000 µg/plate +S9 Negative (1995)
Chromosomal aberration, Cultured human 1-100 µg/ml Positive Dhillon & Dhillon
sister chromatid lymphocytes (1995)
exchange
Micronucleus formation Human cells Positive IARC (1987)
Aneuploidy, chromosomal Human cells Negative IARC (1987)
aberrations, sister
chromatid exchange
Aneuploidy, unscheduled Rodent cells Positive IARC (1987)
DNA synthesis
Mutagenicity, DNA damage, Rodent cells Negative IARC (1987)
sister chromatid
exchange
Cell transformation, Syrian hamster 0-10 µg/ml Positive Tsutsui et al.
numerical chromosomal embryo cells (-S9) (1987)
changes
Gene mutation, Syrian hamster 0-10 µg/ml Negative Tsutsui et al.
chromosomal aberration, embryo cells (-S9) (1987)
sister chromatid
exchange, unscheduled
DNA synthesis
Table 11. (continued)
End-point Test system Dose Result Reference
Numerical chromosomal Cultured human 0.05-75 µmol/l Positive Schuler et al.(1998)
changes lymphocytes
Chromosomal breakage Cultured human 0.05-75 µmol/l Negative Schuler et al.(1998)
lymphocytes
DNA damage pBR322 (-S9) 0.01-0.1 mmol/l Single-strand Yoshie & Ohshima
breaks with 2- (1998)
and 4-hydroxy
estradiol and
estrone; negative
with estradiol
and estrone
Microtubule disruption Chinese hamster 0-100 µmol/l EC50, 10 µmol/l Aizu-Yokota et al.
V79 cells (1995)
Adduct formation
Syrian hamster embryo cells 1 µg/ml (-S9) Increase with Hayashi et al.
estradiol and (1996)
2- and 4-hydroxy
estradiol
Syrian hamsters 2-150 mg/kg bw Increase in Han & Liehr
intraperitoneally kidney at 50 (1994a)
mg/kg bw; no
increase in liver
Male Syrian hamsters 50 mg/kg bw Increase in Han & Liehr
intraperitoneally kidney; no (1994a)
time dependence
Table 11. (continued)
End-point Test system Dose Result Reference
Male Syrian hamsters 100 mg/kg bw Increase in liver Han & Liehr
intraperitoneally 1-2 h after (1994a)
dosing but not
later
Male Syrian hamsters 25 mg Increase in Han & Liehr
subcutaneous implant kidney on day 3 (1994a)
but not day 6;
no hepatic
adducts;
substantial
differences in
adduct levels in
controls between
days 3 and 6
Male Syrian hamsters 100 mg/animal per Negative for Han & Liehr
day intraperitoneally kidney with (1994a)
for 3 days estradiol and
2- and 4-hydroxy
estradiol
NBL rats Dose not reported; Unidentified Han et al.
serum level 14 times adduct after 16 (1995)
that of control but not 8 weeks
of treatment
Mongrel dogs Dose not reported Decrease in Winter & Liehr
prostate adduct (1996)
level; increase
in carbonyl
content
Table 11. (continued)
End-point Test system Dose Result Reference
Human liver 2 mmol/l Negative Seraj et al.
(1996)
Rat liver 2 mg/kg bw per day Adducts in male Feser et al. (1996)
by gavage but not for female liver
14 days
Human breast tissue Positive Musarrat et al.
correlation (1996)
Human breast tissue No correlation Nagashima et al.
(1995)
In vivo
Micronucleus formation, Mouse bone marrow 100-10 000 µg/kg Positive at Dhillon & Dhillon
sister chromatid exchange bw as single highest dose (1995)
intraperitoneal
injection
Micronucleus formation Rat bone marrow 20 µg/kg bw as Negative Ashby et al.
three daily (1997)
subcutaneous
injections
Micronucleus formation Mouse bone marrow 10-10 mg/kg bw Negative Ashby et al.
as single (1997)
intraperitoneal
injection
Micronucleus formation Mouse and rat bone 0.1-10 mg/kg bw Negative Shelby et al.
marrow intraperitoneally (1997)
Table 11. (continued)
End-point Test system Dose Result Reference
Frequency of Male and female mice 310-1250 mg/kg bw Negative Shelby et al.
polychromatic as three (1997)
erythrocytes injections
Male Syrian hamster 5-150 mg/kg bw Negative Han & Liehr
intraperitoneally (1994a)
DNA damage Male Syrian hamster kidney 25 mg subcutaneously Single-strand Han & Liehr
and liver every two weeks breaks in kidney (1994a)
but not liver
DNA damage Male Syrian hamster 250 µg/animal per Single-strand Han & Liehr
day for 7 days by breaks with (1994a)
infusion estradiol and
4-hydroxy but
not 2-hydroxy
Chromosomal aberration Male Syrian hamster 20 mg via Positive in Banerjee et al.
subcutaneous capsule kidney (1994)
DNA damage NBL rat Subcutaneous capsules, Single-strand Ho & Roy (1994)
16 weeks; dose breaks in
not reported prostate with
estradiol +
testosterone
The effect of estradiol at 0.05-75 µmol/L was examined in
cultured human lymphocytes by multicolour fluorescence in-situ
hybridization. DNA probes for the centromere and adjacent
heterochromatin regions of chromosomes 1, 9, and 16 were used to
detect hyperdiploidy, polyploidy, and chromosomal breakage. Nonlinear
increases in hyperdiploidy but no chromosomal breakage was observed.
The authors concluded that induction of numerical changes in
chromosomes by estradiol followed a sublinear dose-response
relationship, probably with a threshold concentration. Binding of
estradiol to microtubules or saturation of detoxification mechanisms
are possible explanations for the observation (Schuler et al., 1998).
Male Syrian hamsters were treated with intraperitoneal injections
of 5, 15, or 150 mg/kg bw estradiol; subcutaneous implants containing
25 mg estradiol for two weeks; or continuous infusion of estradiol or
2-or 4-hydroxy-estradiol at 250 mg/animal per day for seven days. A
single intraperitoneal injection of estradiol had no effect on DNA
single-strand breaks in liver or kidney DNA, but the subcutaneous
implants increased the number of renal single-strand breaks by 10%; no
effect was seen in liver. Infusion of estradiol or 4-hydroxyestradiol,
but not 2-hydroxyestradiol, for one week resulted in a 9% increase in
the number of single-strand breaks relative to untreated controls. The
authors suggested that estrogen-induced carcinogenesis is mediated by
free-radical damage (Han & Liehr, 1994a).
Male Syrian hamsters received capsules containing 20 mg
diethyl-stilbestrol, estradiol, moxestrol, 17 alpha-estradiol, or
ß-dienestrol, and between 94 (dienestrol) and 140 (diethylstilbestrol)
µg/day were obsorbed daily from the pellet. Animals were sacrificed at
0.5, 1, 2, 3, 4, or 5 months (diethylstil-bestrol) or at 5 months (all
other treatments). Chromosomal aberrations but not exchanges in
hamster kidney DNA were cumulative with continued exposure to
diethylstilbestrol. The kidneys of estradiol-and moxestrol-treated
animals had chromosomal aberrations at frequencies similar to those
seen with diethylstilbestrol, whereas the frequency of chromosomal
aberrations in animals treated with the weaker estrogens were similar
to those of controls. The authors suggested that estrogen-induced
chromosomal aberrations are involved in tumorigenesis but that the
process does not involve metabolic activation, since moxestrol, which
is poorly metabolized, did induce chromosomal aberrations (Banerjee et
al., 1994).
2-Catechol estradiol and 4-catechol estrone at 0.01-0.1 mmol/L
induced strand breaks in the pBR322 plasmid, and the level was greatly
enhanced by a nitric oxide-releasing compound. The strand breaks could
be inhibited by antioxidants such as N-acetylcysteine and ascorbate
and by superoxide dismutase. Estradiol, estrone, O-methylcatechol
estrogens, and diethylstilbestrol did not induce strand breaks. The
authors suggest that NO mediates conversion of catechol estrogens to
quinones, and the oxygen radicals produced by the quinone/hydroquinone
redox system react with NO to form peroxynitrite, which causes strand
breaks (Yoshie & Ohshima, 1998).
Natural estrogens and their derivatives were tested for the
ability to induce microtubule disruption in Chinese hamster V79 cells
(which lack cytochrome P450 enzymes) at concentrations of 1-100
µmol/L. The EC50 values were 10 µmol/L for estradiol and 9 µmol/L for
17 alpha-estradiol. The most potent disrupting agent tested was
2-methoxyestradiol (EC50, 2 µmol/L). Preincubation of cells with 1
µmol/L taxol for 2 h protected them against microtubule disruption by
estradiol at doses up to 50 µmol/L. The authors concluded that some
natural estrogens cause microtubule disruption in a nongenomic manner
(Aizu-Yokota et al., 1995).
An increase in the frequency of DNA strand breakage and
accumulation of lipid peroxidation products in the dorsolateral but
not the ventral prostate were seen in four NBL rats treated
subcutaneously with testosterone plus estradiol, relative to control
rats. Treatment of castrated rats with testosterone resulted in a
slightly lower rate of strand breaks than in untreated controls. The
authors concluded that estradiol was responsible for the single-strand
breaks in these animals (Ho & Roy, 1994).
Studies of adducts
Male Syrian hamsters were injected intraperitoneally with 2, 10,
50, or 150 mg/kg bw estradiol and sacrificed 4 h later; with 50 mg/kg
bw and sacrificed 1-8 h later; or with 100 mg/kg bw and sacrificed 1-8
h later. Their livers and kidneys were examined for
8-hydroxy-2'-deoxyguanosine (8-OHdG) as a marker of hydroxy radical
interaction with DNA. Four hours after dosing with 50 mg/kg bw, the
renal 8-OHdG levels were double those of controls; adducts were not
determined in kidneys from animals treated at 150 mg/kg bw. No
dose-dependence was observed. The levels of hepatic DNA adducts in
treated animals were similar to those in controls. In hamsters treated
with 50 mg/kg estradiol and killed 1-8 h later, the level of renal DNA
adducts was greater than that in controls at 4 h but not at 1, 2, or 8
h after dosing; hepatic DNA adducts were not determined. In animals
injected with 100 mg/kg estradiol, the number of hepatic DNA adducts
was increased 1 and 2 h after dosing. Treatment of hamsters with
subcutaneous implants containing 25 mg estradiol for three or six days
increased the renal levels of 8-OHdG by 50% over that in controls by
day 3, but the levels were no different from those of controls in
animals implanted with estradiol for six days. No effect was observed
on the background level of liver DNA adducts at either time. Treatment
of hamsters for three days by intraperitoneal injection with 100
µg/animal per day estradiol or 2-or 4-hydroxyestradiol also had no
effect on renal DNA 8-OHdG levels. The authors concluded that the
mechanism of the carcinogenic action of estrogen occurs through
generation of free radicals via redox cycling of catechol estrogen
metabolites (Han & Liehr, 1994b). Substantial differences were
observed in the levels of adducts in untreated animals after three and
six days in the implant experiment. While no statistical analysis was
performed, the differences were sufficient to indicated a substantial
variation in the background level of adducts. The lack of dose-and
time-dependence of adduct formation in these experiments is
inconsistent with the hypothesis that estradiol is a genotoxic
carcinogen.
An unidentified adduct was observed in DNA isolated from the
dorsolateral prostate of NBL rats treated by subcutaneous
administration of estradiol and testosterone for 16 weeks but not 8
weeks. The circulating estradiol concentrations were increased
approximately 14 times over those of controls, while normal plasma
testosterone concentrations were maintained. The appearance of the
adduct correlated with the appearance of dysplastic lesions (Han
et al., 1995).
Incubation of Syrian hamster embryo cells with 1 µg/ml estradiol
or 2-or 4-hydroxy estradiol for 24 h induced DNA adduct formation in
parallel with cell transformation. The level of DNA adduct formation
was greatest with 4-hydroxy estradiol and then with 2-hydroxy
estradiol and estradiol. No exogenous metabolic activation was used in
these experiments. In later experiments, diethylstilbestrol increased
adduct formation in the absence but not in the presence of metabolic
activation (Hayashi et al., 1996).
Mongrel dogs were treated with capsules containing 5
alpha-dihydro-testosterone (DHT) and/or estradiol for 60 days. The
capsules were of uniform length (7 cm), but the quantity of hormone
used was not described. Blood sampoles were obtained for the
measurement of hormone. The 8-OHdG adduct levels in DNA from prostate
were reduced in all dogs that received DHT, whereas treatment with
estradiol or DHT plus estradiol had no effect. Free radical-induced
damage (carbonyl content) of proteins was observed in prostate tissue,
and the authors concluded that the damage was consistent with injury
by estrogen metabolites followed by DHT-stimulated growth of altered
prostatic cells (Winter & Liehr, 1996).
Sterol-initiated DNA adduct formation was examined in vitro by
32P-postlabelling. After exposure of human liver DNA to 2 mmol/L
steroid, several steroids but not estradiol, estrone, or estriol
formed DNA adducts. The presence of a carbonyl group at C17 (which
estradiol lacks) was strongly associated with DNA binding (Seraj et
al., 1996).
When three male and three female Han:Wistar rats given estradiol
at 2 mg/kg bw per day intragastrically as an aqueous microcrystalline
suspension for 14 days, an estrogen-specific DNA adduct was observed
by 32P-postlabelling in the livers of male but not female rats (Feser
et al., 1996).
To assess the hypothesis that estrogen-induced adduct formation
is related to estrogen-induced tumorigenesis in humans, DNA from
normal human breast tissue, benign tumours, and malignant tissue with
invasive ductal carcinoma was examined for the presence of 8-OHdG
adducts by a novel solid-phase immunoslot-blot assay with
adduct-specific antibodies. The amounts of 8-OHdG found in the three
tissues were 0.25, 0.98, and 2.4 pmol/µg DNA, respectively; 13 times
more endogenous formation of 8-OHdG was observed in MCF-7 cells which
undergo hormone-dependent cell growth and have estrogen receptor s
than in normal cultured human mammary epithelial cells, but no
difference in adduct levels was observed between normal cells and
MDA-MB 231 cells, which undergo receptor-independent growth and lack
estrogen receptors. 8-OHdG adduct levels also correlated to the
estrogen receptor status of the tissue, with higher adduct levels in
malignant tissue with estrogen receptors than in those without. Age
and smoking status did not correlate to the 8-OHdG content of DNA. The
authors concluded that accumulation of 8-OhdG adducts in DNA is
predictive of the risk for breast cancer and may be a major
contributor to the development of breast neoplasia (Musarrat
et al., 1996).
No difference in 8-OhdG adduct levels was found by
high-performance liquid chromatography-electrochemical detection in
breast cancer tissue and adjacent non-cancerous tissue, and no
correlation was found with expression of estrogen or progesterone
receptors or with clinical stage or histological grade (Nagashima et
al., 1995).
1.3.5 Reproductive toxicity
The embryotoxicity and teratogenicity of estradiol were reviewed
by a working group convened by IARC (1979), which concluded that
"Oestradiol-17ß has teratogenic actions on the genital tract and
possibly on other organs and impairs fertility."
Estradiol was administered in the feed to female Crl:CD BR rats
at doses equal to 0, 0.003, 0.17, 0.69, or 4.1 mg/kg bw per day in a
90-day, one-generation study. As no pups were born to dams at the two
highest doses, only three dose groups of the F1 generation were
assessed. The mean daily intakes of the F1 females were 0, 0.005 and
0.27 mg/kg bw per day, respectively. The F0 rats were 49 days of age
on test day 0, and serum hormones were evaluated after 7, 28, and 90
days of feeding; they were evaluated on postnatal day 98 for the F1
generation. In the F0 generation, estradiol at doses > 0.17 mg/kg bw
per day produced a dose-dependent increase in serum estradiol
concentration, and all doses produced a dose-dependent decrease in
serum progesterone concentration on test day 90, which correlated with
ovarian atrophy and lack of corpora lutea. The serum concentration of
luteinizing hormone was decreased at all times at > 0.69 mg/kg bw
per day and at 0.17 mg/kg bw per day on test day 90. Little change was
observed in the serum concentrations of follicle-stimulating hormone.
The serum concentration of prolactin was increased at 4.1 mg/kg bw per
day on test day 90. In the F1 generation on postnatal day 28, the
serum estradiol concentration was increased and that of progesterone
decreased at 0.27 mg/kg bw per day. No change in serum prolactin,
follicle-stimulating hormone, or luteinizing hormone concentration was
noted. Dietary estradiol caused marked effects on the estrus cycle at
0.17 mg/kg bw per day (F0) and 0.27 mg/kg bw per day (F1) and at
0.69 and 4.1 mg/kg bw per day (F0 generation) (Biegel et al., 1998b).
Information on the pups in this study was presented elsewhere.
The groups at the two highest doses produced no pups, and the weights
of the pups in the two remaining groups decreased relative to that of
controls; the weights of pups of the F0 generation at 0.003 mg/kg bw
per day (0.005 mg/kg bw per day for the F1 generation) recovered
after birth and remained similar to those of controls throughout the
study. The mean length of gestation in this dose group was
statistically nonsignificantly decreased, which the authors suggested
contributed to the decreased birth weight. The body weights of animals
at 0.27 mg/kg bw per day remained below control levels throughout the
study. Parenteral administration of estradiol did not affect the
anogenital distance in male or female pups. Onset of sexual maturity,
as measured by prepubertal separation in males and vaginal opening in
females, was delayed. Some of the histopathological findings were more
severe in the F1 generation than in the parent generation. The
authors concluded that additional studies were needed to define the
dose-response curve more accurately (Biegel et al., 1998a).
The average litter size of transgenic 'knockout' female mice
deficient in steroid 5 alpha-reductase type I (SRD5 alpha 1-/-,
wild-type C57Bl/6J/129Sv) is reduced in comparison with wild-type
controls (2.7 vs. 8 pups, respectively). In reductase-deficient
animals, the maternal serum estrogen concentrations were chronically
increased by two-to threefold relative to control animals. In control
animals, spikes in DHT and to a much smaller extent testosterone
concentrations occurred in maternal plasma on day 9 of gestation. In
the 5a-reductase-deficient animals, the androgen peaks at day 9 were
reversed. Oogenesis, fertilization, implantation, and placental
morphology appeared normal in reductase-deficient animals. Fetal loss
occurred between gestation days 10.75 and 11, commensurate with a
surge in placental androgen production. Minimal fetal loss was
observed on gestation day 10.5. To test the hypothesis that steroid
hormones contribute to fetal loss in reductase-deficient animals,
pregnant animals were treated with pellets containing various amounts
of steroid hormone. Table 12 shows the effect on mean embryo survival.
Bleeding was observed grossly in the uteri of control and experimental
animals. Administration of estrogen receptor antagonists or inhibitors
of aromatase prevented the excess fetal loss observed in the
reductase-deficient mice. Testosterone was mildly protective against
fetal loss in the knockout mice. The authors concluded that
5a-reductase guards against the toxic effects of estrogen during
pregnancy. They also noted that the human placenta, unlike the rodent
placenta, has high levels of aromatase, resulting in very high
concentrations of estrogens in the amniotic fluid. They speculated
that the human fetoplacental unit has developed a mechanism to protect
itself against estrogens (Mahendroo et al., 1997).
Pregnant female CF-1 mice were treated subcutaneously on
gestation day 13 with Silastic capsules containing 0, 25, 100, 200, or
300 µg estradiol. Male fetuses positioned in utero between a male and
female (MF males) were examined for treatment-related prostatic
effects. In some experiments, MF males were obtained from pregnant
dams killed on gestation day 19 and reared by foster dams. At the age
of seven months, MF males were castrated, implanted subcutaneously
with capsules containing 500 µg testosterone, and killed three weeks
later. The total concentration of estradiol in serum was increased in
a dose-dependent manner in treated MF fetuses collected on day 18,
with 94, 150, 230, 360, and 530 pg/ml in the animals at the five
doses, respectively. A 40% increase in the number of prostatic
glandular epithelial buds was found in MF males from dams treated with
25 µg estradiol, relative to controls. An increase in prostate size
was also noted, but the size of the individual buds was not changed;
prostate weight was increased in MF males at the low dose sacrificed
at eight months but was decreased at the high dose, resulting in an
inverted-U dose-response curve. The authors concluded that increased
fetal serum estrogen concentrations affect androgen regulation of
prostate differentiation, resulting in a permanent increase in the
number of prostatic androgen receptors and in prostate size (vom Saal
et al., 1997). The doses used in this study were below the NOEL.
Under conditions associated with reduced estrogen synthesis in
humans (aromatase deficiency, placental sulfatase deficiency, fetal
anencephaly), estrogen production and concentrations may be reduced by
80-90%. However, progesterone production and fetal development remain
normal, indicating that considerably more estrogen is produced during
normal pregnancy than is necessary (Fisher, 1998).
The embryotoxicity of steroidal and nonsteroidal estrogens was
examined in cultured whole embryos obtained from Sprague-Dawley rats.
Preliminary experiments resulted in steep dose-response curves for all
estrogens examined at doses ranging from 0.05 to 0.5 mmol/L. For
example, diethylstilbestrol had no effect at concentrations < 0.15
mmol/L but was lethal to 100% of cultured embryos at doses > 0.25
mmol/L. The concentrations tested resulted in low embryolethality
(2-20%). Estrogens had dysmorpho-genic effects at concentrations of
0.1-0.2 mmol/L. The commonest effect observed was hypoplasia of the
prosencephalon. Estradiol and estrone were markedly and statistically
significantly more toxic in the presence of metabolic activation (from
livers of pregnant and non-pregant females and Aroclor 1254-treated
adult male rats), but metabolic activation attenuated the embryotoxic
effects of ethinylestradiol, tamoxifen, and erythrohexestrol and had
no effect on other estrogens. In this system, estradiol was more
efficiently converted to catechol estrogens in male liver, but
Table 12. Effects of steroid hormones in 21-day release pellets on embryo survival
Pellet Dose Control females Srd5a1-/- females
treatment
No. Live Dead No. Live Dead
litters embryos embryos litters embryos embryos
None 4 8.0 0.4 8 3.2 5.1
Placebo 3 9.6 0.67 3 4.3 5.0
Androstenedione 0.5 4 3.3 4.5 5 1.0 7.8
Testosterone 0.5 2 8.8 1.5 5 6.2 3.2
Estradiol 0.5 2 0 7.0 5 0 8.4
0.08 5 0 7.4 6 0 7.8
0.02 2 0 5.0 6 0.3 6.3
0.01 2 0 11 6 0 6.8
0.005 2 0 8.5 5 0 5.0
0.0025 2 4.5 4.0 4 2.8 5.5
ethinylestradiol was converted to catechol estrogens approximately
three times more effectively than estradiol when metabolic activation
systems from pregnant and non-pregnant animals was used. The authors
concluded that the effects observed are independent of steroid
structure or estrogen activity and are strongly dependent on the
pathways and rates of biotransformation of some (but not all) of the
parent compounds (Beyer et al., 1989).
Ten mg of estradiol or testosterone were implanted subcutaneously
into groups of five and seven female Sprague-Dawley rats on day 10 of
pregnancy, whereas control pregnant rats were given dextran by the
same method. Implantation with estradiol or testosterone resulted in
complete resorption of embryos in all treated animals (Sarkar et al.,
1986).
A review of the birth certificates and hospital records of 7723
infants whose mothers had reported using oral contraceptives indicated
that these compounds present no major teratogenic hazard (Rothman &
Louik, 1978).
Studies of reproductive toxicity are summarized in Table 13.
1.3.6 Special studies of mechanisms of action
Estrogen-induced tumorigenesis has been the subject of two lines
of investigation: receptor-based effects and redox cycling and DNA
adduct formation leading to genetic damage. During the past decade,
considerable attention has been focused on understanding the molecular
basis of hormone receptor biology. Recently, transgenic animals that
overexpress or lack estrogen receptors (Couse et al., 1997b) or
aromatase (Fisher et al., 1998) have been developed. The role of
estrogens and other hormones in mammary neoplasia in rodents and their
relevance to human risk has been reviewed (Russo & Russo, 1996), and
it was noted that rodent models mimic some but not all of the complex
external and endogenous factors involved in initiation, promotion, and
progression of carcinogenesis. Tumour type and incidence are
influenced by the age, reproductive history, and the endocrine milieu
of the host at the time of exposure. The spontaneous incidence of
tumours differs in different strains of rats and mice. In rats, most
spontaneously developed neoplasias, with the exception of leukaemia,
are of endocrine organs or organs under endocrine control. Russo &
Russo (1996) concluded that mechanism-based toxicology is not yet
sufficient for human risk assessment, and the approach should be
coupled to and validated by traditional long-term bioassays.
The estrogen-responsive male Syrian hamster kidney model has been
widely used to study the carcinogenicity of estrogens in vivo.
Separation of carcinogenic from hormonal effects in male and
ovariectomized female Syrian hamster kidney has been reviewed (Yager &
Liehr, 1996). In hamsters treated chronically with relatively high
doses by subcutaneous implantation, certain potent synthetic estrogens
such as ethinylestradiol result in < 10% tumour incidence in kidney,
whereas treatment with other estrogens result in renal tumour
development in nearly all animals. Ethinylestradiol also acts at
different sites from other estrogens in the hamster kidney. Thus, the
estrogenicity of a compound is essential but not sufficient for renal
carcinogenesis in hamsters. Other factors that have been suggested to
contribute to carcinogenicity include cell-type-specific uptake and
differential estrogen metabolism (such as high 4-hydroxylation rates)
leading to estrogen-induced damage to cell macro-molecules (DNA
and protein). Similarly, the progesterone metabolite
5 alpha-pregnane-3,20-dione successfully competes with progesterone
for receptor binding and biological effectiveness in some tissues but
not others (Tsai et al., 1998). Since a second estrogen receptor
isoform (ERb) has recently been identified, the results described
below should be interpreted with caution.
The estrogen metabolite 4-hydroxyestradiol, but not
2-hydroxyestradiol, was tumorigenic in male hamster kidneys (Yager &
Liehr, 1996), the proposed mechanism of action being redox cycling
resulting in oxygen radical formation and subsequent damage to cell
macromolecules. It is not certain that this pathway is relevant
in vivo at physiological concentrations of estradiol. For example,
micromolar concentrations of estradiol are necessary to cause
microtubular disruption in Chinese hamster V79 cells, and these are
greatly in excess of the picomolar to nanomolar concentrations
normally found in serum. At higher concentrations, the lipophilicity
of estradiol and some metabolites (such as methoxy derivatives) and
their ability to intercalate into DNA and lipid membranes may be more
important from a toxicological perspective than the estrogenic
properties.
The hormonal (i.e. receptor-mediated) and carcinogenic (i.e.
genotoxic) properties of synthetic hormones were differentiated by
measuring the rates of catechol estrogen and methyl ester formation by
a weak carcinogen, 17a-ethinylestradiol, and by a strong hormonal
carcinogen, moxestrol. The rates of hydroxylation in comparison with
that for estradiol were 40-50% for moxestrol and 25-35% for
ethinylestradiol, the differences being apparent at longer reaction
times (i.e. 20 min but not 10 min). 2-Hydroxymoxestrol was a poor
substrate for COMT, proceeding at a rate of about 3% of the
methylation of 2-hydroxyestradiol. In hamster kidney cytosolic protein
extracts, 10 nmol/L progesterone decreased binding of 2 nmol/L
3H-progesterone by 78%, and 10 nmol/L ethinylestradiol inhibited it
by 35%. Interpretation of this result is complicated, as the assay was
performed with insufficient excess progesterone. Estradiol and
moxestrol had no effect. The authors suggested that the decreased
capacity of ethinylestradiol to form catechol metabolites and its
progestogen antagonist activity contribute to the low tumour incidence
seen with this compound (Zhu et al., 1993).
Table 13. Studies of the reproductive toxicity of estrogens
Species Dose Findings Reference
Mice, rats 0.1-35 mg/day Teratogenic IARC (1979)
subcutaneously
Female rats 0.003-4.1 mg/kg bw No NOEL identified Biegel et al.
per day orally for (1998b)
90 days
Transgenic mice Estrogen No NOEL; effects Mahendroo et
concentrations observed between days al. (1997)
reduced two- 10.75 and 11
to threefold
Mice 0-300 µg/animal No NOEL for effects on vom Saal et
fetal prostate al. (1997)
Cultured whole 0.5-0.5 mmol/l Dysmorphogenic effects Beyer et al.
embryos of at 0.1-0,2 mmol/L (1989)
Sprague-Dawley
rats
Sprague-Dawley 10-mg implant Embryo resorption Sarkar et al.
rats (1986)
Humans Acidental exposure No effect reported Rothman & Louk
(1978)
Several steroidal estrogens were tested at doses of 0.1-100
nmol/L for their ability to increase proliferation of primary renal
proximal tubular cells in culture. Most of the estrogens tested
(including 4-hydroxyestradiol and estrone and, to a lesser extent,
2-hydroxyestradio) increased cell proliferation at a concentration of
0.1-10 nmol/L but inhibited it at 100 nmol/L. The authors concluded
that the ability to induce cell proliferation is a more accurate
predictor of carcinogenicity in this system than estrogen-responsive
end-points or the amount of catechol metabolites generated (Li et al.,
1995).
To determine the role of estrogens in tubular renal damage and
the subsequent reparative cell proliferation, castrated adult male
Syrian hamsters were given subcutaneous pellets that released hormones
at the following rates (µg/day): diethylstilbestrol, 145; estradiol,
134, estrone, 104, ethinylestradiol, 154; tamoxifen, 141;
progesterone, 147; and DHT, 121. Diethylstilbestrol was administered
for one to nine months, while the other compounds were administered
for five months. The severity of tubular damage increased with
progressive estrogen treatment, with a prominent rise in the number of
secondary and tertiary lysosomes. The concentration of cathepsin D was
increased in estrogen-treated kidneys (by approximately 2.7-and
3.5-fold at four and five months, respectively) and paralleled the
rise in estrogen receptor content. Progesterone and DHT alone had no
effect, and concomitant treatment of animals with estrogen and either
tamoxifen or DHT mitigated the estrogenic effects. The primary form of
cathepsin D found in the kidneys of control and estrogen-treated
animals was the 52-kDa isoform, considered to be the inactive form of
the protein. The 31-and 27-kDa isoforms, believed to be the active
forms, were found in significant amounts only in the kidneys of
estrogen-treated animals, primary renal tumours, and their metastases.
The authors suggested that cathepsin D mediates renal tubular damage
as a first step in reparative cell proliferation (Li et al., 1997).
Estradiol- or diethylstilbestrol-induced growth of cultured
proximal renal tubular cells could be inhibited by ethinylestradiol.
Expression of estrogen-responsive protooncogene (c- myc, c- fos, and
c- jun) RNA and protein in kidneys was reduced in animals treated
concomitantly relative to that found in animals treated with estradiol
or diethylstilbestrol. The authors concluded that ethinylestradiol
interferes with estrogen receptor-mediated mitogenic pathways,
preventing gene dysregulation and tumour development. This effect does
not appear to be due to differential binding to estrogen receptors by
estrogenic substances (Li et al., 1998).
Other hormones, notably progesterone, testosterone, and
deoxycortico-sterone, and the antiestrogen tamoxifen prevent or
inhibit the growth of estrogen-induced renal tumours in Syrian
hamsters (reviewed by Yager & Liehr, 1996). Progesterone and tamoxifen
exert a protective effect on mammary carcinogenesis (Inoh et al.,
1985). A review of clinical data indicated that adjuvant progestogen
therapy for treatment of patients with metastatic renal-cell carcinoma
is not effective, indicating that carcinogenesis in the Syrian hamster
model is not representative of human renal carcinogenesis (Linehan et
al., 1997).
Rodent tissues that form estrogen-induced tumours have high
concentrations of the caetcholamine noradrenaline. In a study to test
the hypothesis that hydrogen peroxide formed by monoamine oxidase
deamination of catecholamines provides a source of free radicals, in
addition to that postulated to be provided by metabolic redox cycling
of catechol estrogen intermediates, Syrian hamsters and Sprague-Dawley
rats received 25 mg estradiol in a subcutaneous capsule for two weeks.
Treatment increased monoamine oxidase activity in hamster kidney but
not liver and had no effect on monoamine oxidase activity in rat liver
or kidney. The induction of hamster kidney monoamine oxidase activity
could be prevented by tamoxifen. The authors concluded that
receptor-mediated induction of monoamine oxidase, which deaminates
catecholamines, may increase production of hydrogen peroxide and
hydroxyl radicals, thus contributing to tumour initiation (Sarabia &
Liehr, 1998).
Male Syrian hamsters were treated with quercetin, an inhibitor of
COMT, in order to assess the potentiating effects of this compound on
renal tumorigenesis. All six animals treated with subcutaneous pellets
that released estradiol at 61 µg/day developed kidney tumours, but no
tumours were seen in hamsters treated with quercetin at 0.3 or 3% in
the diet for 5.7 or 6.5 months, respectively. Concomitant
administration of estradiol and quercetin increased the number of
large tumours and the incidence of metastases over that seen with
hormone treatment alone. Quercetin inhibited 2 and 4-catechol estrogen
methylation by 34 and 22%, respectively. The rates of redox cycling in
liver and kidney were not affected by treatment with quercetin or
estradiol (Zhu & Liehr, 1994).
Male Syrian hamsters received subcutaneously implanted pellets
containing 25 mg estradiol (which were replaced every three months)
for seven months. Dietary supplementation with 1% vitamin C decreased
estrogen-induced renal carcinogenesis by 50% in a small number of male
Syrian hamsters. In related experiments, the effect of estradiol
and/or vitamin C was examined on the renal activity of the detoxifying
enzymes quinone reductase, catalase, superoxide dismutase, glutathione
peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase,
and gamma-glutamyl transpeptidase. Glutathione peroxidase activity was
increased in the kidneys of hamsters treated with estradiol for 1
month (141% of control). Quinone reductase activity was reduced in the
kidneys of estradiol-treated animals (18% of control), but the
activity was partially restored by dietary supplementation with
vitamin C for one month (32% of control); in liver, concomitant
treatment with vitamin C and estradiol reduced the activity of this
enzyme (6% of control), and estradiol treatment alone caused a smaller
decrease in activity (68% of control). Differences in catalase
activities were observed after one month but not by seven months of
treatment. Vitamin C had no effect on the intensity or specificity of
estrogen-related kidney DNA adducts. The authors concluded that the
enzymatic changes observed in estradiol-and/or vitamin C-treated
animals were insufficient to account for the differences in renal
tumour incidence. The authors concluded that vitamin C inhibits
estrogen-induced carcinogenesis by reducing the concentration of
estrogen quinone metabolites (Liehr et al., 1989).
COMT is present in the epithelial cells of the proximal
convoluted tubules of the kidney, predominantly in the juxtamedullary
region, where estrogen-induced tumours arise. Treatment of male Syrian
hamsters with estradiol or ethinylestradiol for two or four weeks
altered the intensity, distribution, and subcellular location of
immunoreactivity to COMT. Staining for this enzyme in control animals
was largely of the soluble cytoplasm and nuclear membrane-bound forms,
whereas staining for the soluble form of nuclear COMT was observed in
estrogen-treated animals. No differences were observed between the two
estrogens or between animals treated with estrogen for two or four
weeks; no difference in nuclear location was observed between treated
and control animals. Estradiol-induced renal tumours did not stain for
COMT, and the nuclear signal present in human cells was lacking in
hamster kidney. The authors suggested that a change in the subcellular
distribution of COMT is a protective response to catechol estrogen
metabolic damage to the genome (Weiss et al., 1998).
Male Noble rats were treated with subcutaneous Silastic implants
containing testosterone and/or estradiol or diethylstilbestrol for 16
weeks. In estradiol-treated animals, the plasma testosterone
concentration, determined after three weeks of treatment, was
decreased more than 10-fold (from 4.8 ng/ml to < 0.3 ng/ml), whereas
the estradiol concentration was increased 4.5-fold (from 16 pg/ml to
75 pg/ml). Diethylstilbestrol but not estradiol caused a statistically
significant decrease in body weight, and both hormones decreased the
relative weight of the dorsolateral and ventral prostate and seminal
vesicles plus coagulating glands. The body weights of animals treated
for 16 weeks with testosterone and estradiol were significantly lower
than those of controls, the relative weights of the dorsolateral and
ventral prostate and seminal vesicles plus coagulating glands were
increased, and the testicular weight was decreased approximately
twofold. Multifocal epithelial dysplasia and marked inflammatory
changes were observed in the lateral prostate. No changes were seen in
the morphology of the ventral prostate seminal vesicle, coagulating
gland (anterior prostate), or ampullary gland. Implants of
testosterone plus diethylstilbestrol induced widespread dysplasia in
the ventral prostate and lesser or no ventral prostatic dysplasia. In
explant cultures, animals treated with testosterone plus
diethylstilbestrol or testosterone plus estradiol showed a reduced
ability to convert the 5 alpha,3ß-hydroxysteroid derivative of 3H-DHT
to the more polar 6 alpha-and 7 alpha-hydroxylated derivatives,
resulting in accumulation of 3ß-androstanediol. These metabolic
changes resulted in a threefold (testosterone plus estradiol) or
eightfold (testosterone plus diethylstilbestrol) increase in
accumulation of 3ß-androstanediol in the dysplastic ventral prostate;
no accumulation was observed in the explanted dorsolateral prostate.
In animals treated with testosterone plus diethylstilbestrol, the
ratio of estrone:estradiol was reversed in the ventral prostate,
whereas in animals treated with testosterone plus estradiol, estradiol
metabolism was decreased in the dysplastic dorsolateral prostate but
not in the ventral prostate. The authors concluded that differences
between target tissues in the bioavailability of the estrogen
component determines in which lobe prostate dysplasia develops (Ofner
et al., 1992).
Male Noble rats were treated with Silastic implants containing
testosterone and estradiol for 16 weeks since it had been reported
previously that such implants increase the plasma estradiol
concentration threefold while maintaining testosterone at
physiological concentrations. This treatment regimen produced
dysplasia in the dorsolateral prostate, without liver dysplasia.
Microsomes were prepared from the liver, ventral prostate, and
dorsolateral prostate of control and treated animals to determine
metabolic conversion of estradiol to catechol estrogens. Catechol
estrogen formation was observed at high levels in liver microsomal
incubates and low levels in prostate incubates. Treatment failed to
alter the extent or profile of hepatic estradiol metabolism, except
for a significant reduction in estriol production relative to
controls. A nonsignificant reduction in 2-hydroxyestradiol formation
was also observed. The authors concluded that catechol estrogen
formation is not a mediating step in estrogen-induced tumorigenesis
(Lane et al., 1997).
Mongrel dogs were treated for 60 days subcutaneously with DHT
and/or estradiol; however, the quantity of hormone in the implant and
the resulting plasma concentrations were not measured. Previous
studies had indicated that such implants maintain plasma hormone
concentrations at physiological levels. The activities of aryl
hydrocarbon hydroxylase, 7-ethoxycoumarin O-deethylase, and
estradiol 2-and 4-hydroxylase were elevated in the prostate glands of
animals treated with either hormone or their combination and were
either decreased or unchanged in liver and kidney. The increase
observed in estradiol-treated animals was substantially modified by
concomitant treatment with DHT. The activity of estrogen 2-hydroxylase
was increased tenfold and fourfold in animals given estradiol and
estradiol plus DHT, respectively. The activities of
7-ethoxycoumarin- O-deethylase, aryl hydrocarbon hydroxylase,
glutathione peroxidase I, and catalase were also increased in the
prostate. Hormones had variable effects on these enzymes in liver and
kidney. The free radical-generated carbonyl content of the prostate
increased 2.5-fold after treatment with estradiol and twofold with
treatment with estradiol plus DHT. No hormone-related effects on
carbonyl content were seen in kidney proteins, whereas DHT and the
combination with estradiol increased the carbonyl content by 60 and
150% over the control level. Treatment with estradiol alone resulted
in a substantial but nonsignificant decrease in the hepatic protein
carbonyl content relative to controls. In DNA hydrol