A Review on the Genotoxic Effects of Some Synthetic Progestins
The present review gives the details of the genotoxic
studies carried out till date for some selected synthetic progestins.
Mutagenicity is defined as a permanent change in content or structure
of the genetic material of an organism. A mutagenic hazard can be manifested
as a heritable change resulting from germ-line mutations and/or somatic
mutations leading to cancer or other chronic degenerative processes such
as aging. Reactive Oxygen Species (ROS) generated through normal metabolic
processes or from toxic products, can lead to a state of oxidative stress
that contributes to the pathogenesis of a number of human disease by damaging
lipids, protein and DNA. Oral contraceptives have been used since the
early 1960s and are now used by about 90 million women world wide. The
pill is given as a combination of an estrogen and a progestogen. The estrogen
component of combined oral contraceptives is either ethinylestradiol or
mestranol and the progestogens used are cyproterone acetate, desogestrol,
ethynodiol diacetate, levonorgestrel, lynestrenol, megestrol acetate,
norethisterone, norethisterone acetate, norethynodrel, norgestimate and
norgestrel. Little is known about the long term health risks and potential
protective effects of these individual components. Synthetic progestins
induced the genotoxic damage and also various types of cancers, both singly
as well as in combination with estrogens. Various synthetic progestins
have been tested for their genotoxic effects in different experimental
models, using different genotoxic end points. Ethynodioldiacetate, norethynodrel,
norgestrel, lynestrenol and medroxyprogesterone acetate were found to
be genotoxic only in the presence of metabolic activation supplemented
with NADP. Megestrol acetate, cyproterone acetate and chlormadinone acetate
were found to be genotoxic in the absence of metabolic activation. On
the basis of reports available it is suggested that the progestins in
which double bond between carbon-6 and carbon-7 is present, they undergo
nucleophilic reaction and generates free radical in the system to show
the genotoxic effects and the progestins in which double bond between
carbon-6 and carbon-7 is absent, they need metabolic activation like estrogens,
such as estradiol-17β and ethinylestradiol to show the genotoxic
Steroid hormones are the members of lipid compounds. They are mainly
secreted by adrenal cortex of mammals, testis in male, ovary in the female
and the placenta of mammals. Steroids consist of a tetracyclic nucleus,
which is named cyclopentanoperhydro-phenanthrene (Fig. 1).
The phenanthrene portion of the nucleus is comprised of rings A, B and
C, while D is the cyclopentane portion. Steroid hormones are responsible
for a number of physiological and pharmacological effects in humans and
other mammals. There are four basic classes of steroid hormones (Gorbman
et al., 1983).
|| Steroid nucleus
Adenocorticoids: Adrenocorticoids are grouped into the (i) Glucocorticoids:
Cortisol, corticosterone and cortisone. They facilitate the formation
of carbohydrates from non-carbohydrate sources (gluconeogenesis) and (ii).
Mineralocorticoids: They have ability to affect water and electrolyte
metabolism (favoring retention of Na+ and excretion of K+),
Estrogens: Estrogens have the ability (secreted by the ovarian
follicle) to stimulate female secondary sex characteristics and to help
maintain the female reproductive tract e.g., estrone, estradiol and estriol.
Progestins: They stimulate the uterus and maintain uterine development
during pregnancy (secreted by the corpus luteum of the ovary) e.g., Progesterone,
Androgens: They stimulate male characteristics and maintain male
sex accessory glands and ducts (secreted by testis) e.g., testosterone,
5 α-dihydrotestosterone and androstenedione. The usefulness of above
classification scheme is limited to some mammalian species, because glucocorticoids
stimulates aspects of female reproduction on in bony fishes. Aldosterone
is usually classified as a mineralocorticoid, but it is a potent glucocorticoid
in some animals.
Structure and nomenclature of steroids: The basic nucleus of steroids
has 17 carbon atoms as shown in the Fig. 2.
The nucleus has six asymmetric carbon atoms, shown with asterisk in the
Fig. 2. The nucleus is a flat structure lying in the
plane of the page. If the hydrogen projects towards the observer, it is
said to be in the cis or-β position (solid line) and if the hydrogen
project away from the observer, it is said to be in the trans or α-position
(broken lines) as shown below:
|| Structure of steroids
Additional carbons can be attached to the 17-carbon nucleus to form other
classes of steroid compounds. If a methyl atom is attached to carbon 13,
a new compound known as estrone (C-18) is formed. If a second methyl group
is added to carbon 10, androstane (C-19) is formed.
Metabolism of steroids: Ovary, testis, adrenal gland and placenta
have the ability to produce steroidal hormones, which is favoured by the
trophic hormones such as andrenocorticotrophic Hormone (ACTH), Luetinizing
Hormone (LH), Follicle Stimulating Hormone (FSH) and Progesterone Releasing
Hormone (PRL) and its receptor in the target tissues. It is also possible
that one steroid secreting organ can take up steroid originally secreted
by another organ and modified its activity. For example, mammalian adrenal
cortex can take up progesterone secreted by the ovary and modifies it
to corticoids. Chemical modifications may also occur in the brain, skin,
salivary glands and other tissues. Steroid hormones are synthesized from
a C27 precursor steroid, cholesterol. There are multiple sources
of this steroid hormone precursor: de novo synthesis within the
endocrine organ and uptake from the circulating pool that is maintained
by the liver or supplied in the diet. In the blood cholesterol circulates
in the form of cholesterol esters bound to Low and High Density Lipoprotein
(LDL and HDL). For the synthesis of the steroid hormone the breaking off
of the six carbon side chain of the cholesterol occurs in mitochondria
(Gorbman et al., 1983).
The inner membrane of the mitochondria contains an oxygenases known as
cytochrome P450, which breaks the side chain of the cholesterol and results
in the formation of pregnenolone (C21). Pregnenolone is further
transported to endoplasmic reticulum, where it is either oxidized to form
progesterone or hydroxylated to form 17-hydroxypregnenolone. Further with
the help of hydroxylating enzyme, desmolase and various cofactors triphosphopyridine
nucleotide (TPNH) and molecular oxygen give rise to various steroid hormones
(Fig. 3). The catabolism of steroid occurs in both steroidogenic
and peripheral tissues. The major site of catabolism is the liver although
the kidney and other tissues may catabolize circulating steroids to some
extent. The major catabolic enzymes are involved in reductive reactions,
particularly in the ring A and carbons 3 and 20. They are reduced to more
water soluble and in active steroid sulfates and glucoronides that are
eliminated through the urine or through the bile fluid, with ultimate
excretion in the feaces (Gorbman et al., 1983).
|| Biogenesis of steroid hormones
Action of Birth Control Pills (BCPs): Normally the pituitary glands
produces two hormones called Follicle Stimulating Hormone (FSH) and Leutenizing
Hormone (LH). These hormones serve to stimulate the ovary to produce an
egg at each menstrual cycle (to ovulate). The ovary is also the production
site for the woman`s two central female hormones estradiol and progesterone,
(Progestin) (Elstein et al., 1976). Birth Control Pills (BCPs)
are a combination of synthetic estrogen and progestin. BCPs fool the pituitary
gland so that it produces less FSH and LH. By reducing the FSH and LH
required for ovulation, birth control pills suppress, but do not eliminate
ovulation. BPCs may have two main effects (Wolf et al., 1979; Chang
and Hunt, 1970): (i) They thin the inner lining of the uterus (called
the endometrium) depleting it of glycogen and blood supply, (ii) BCPs
may thicken the cervical mucus, making it more difficult for the sperm
to travel up through the cervix. Intergrins are a group of adhesion molecules
that have been implicated as playing an important role in fertilization
and implantation. According to Somkuti et al. (1996) there are
several types of integrins and it is believed that the endometrium is
most receptive to implantation when it expresses certain types of integrins.
BPCs change the type of integrins that the endometrial lining produces
and makes the implantation difficult of the pre-born child (Somkuti et
Genotoxic damage and cancer risks of oral contraceptives: The
type of oral contraceptives prescribed differs between countries and both
the type of oral contraceptive and the doses of estrogens and progestogens
have changed between and within countries overtime (Joosten et al.,
2004). Chromosomal abnormalities and sister chromatid exchanges have been
reported in peripheral blood lymphocytes of women taking oral contraceptives
(Carr, 1967, 1970; Goh, 1967; Murthy and Prema, 1979). There is sufficient
data which indicates that the prolonged use of the synthetic progestins
can cause cancer among animals and humans (Rudali, 1975; El-Etreby and
Gräf, 1979; Misdorp, 1991; Deml et al., 1993; Martelli et
al., 1996; Heinemann et al., 1997; Maier and Herman, 2001).
Synthetic progestins have a wide spread use in medicine but their side
effects are often debatable. Due to the genotoxic hazards and carcinogenic
risks of synthetic progestins it is important to review the studies carried
out till date. The present review is limited to some commonly use synthetic
progestins in oral contraceptives formulations. Progestins were first
isolated in 1993 and progesterone itself was synthesized in the 1940s.
There are different classes of progestins, such as progesterone, retroprogesterone,
progesterone derivative, 17α-hydroxy progesterone derivatives (pregnanes),
17α-hydroxy nor progesterone derivatives (non-pregnanes), 19-norprogesterone
derivatives (nor pregnanes), 19-nortestosterone derivatives (estranes),
19-nortestosterone derivatives (gonanes) and spirolactone derivative.
Schindler et al. (2003) has classified progestins in nine groups
(Table 1) and the details for the genotoxic studies
carried out till date is given in Table 2.
|| Types of Progestins
|| Genotoxicity studies for synthetic progestins carried
out till date
Medroxy progesterone acetate (CAS: 71-58-9)
Colour: White or off-white
Solubility: Medroxyprogesterone acetate is practically insoluble
in water, sparingly soluble in alcohol and in methyl alcohol, slightly
soluble in ether freely soluble in chloroform, acetate, dioxin, dimethylsulphoxide.
Postulated mode of action: Medroxy progesterone acetate (Fig.
4) is a progestin that is derived from the naturally occurring female
hormone, progesterone. It is the derivative of 17α-hydroxyprogesterone
(Pregnanes). It is used to treat abnormal uterine bleeding, promote menstrual
cycles and to treat symptoms of the menopause. Medroxyprogesterone is
use to promote menstruation when women do not begin naturally to menstruate
at puberty (called primary amenorrhea) or if they stop menstruating before
menopause (called secondary amenorrhea). It is used in the treatment of
endometrial, prostate and renal cancer. It is also used for treating abnormal
bleeding from the uterus in many situations. It is also used in combinations
with estrogens for treating symptoms of menopause in order to prevent
unchecked growth of the endometrium that may lead to endometrial cancer.
It is secreted in breast milk, but the effect on infant has not been determined
(Gilstrap and Little, 1992; Briggs et al., 2005). After oral intake
medroxyprogesterone does not undergo any first pass effect. The bioavailability
is 100%. It has no binding affinity to Sex-Hormone Binding Globulin (SHGB)
and Corticosteroid Binding Globulin (CBG) and in serum medroxy progesterone
acetate is bound to albumin for 88%. It is extensively metabolized in
the liver (Schindler et al., 2003).
|| Medroxy progesterone acetate
Cancer studies: Medroxyprogesterone acetate causes reversible
changes in the endometrium, from proliferative to secretary or suppressed.
Medroxyprogesterone acetate induced adeno carcinomas of the mammary gland
in female mice and malignant mammary tumors in dogs. In female dogs a
dose-related increase in the incidence of large mammary nodules was also
found after intra-muscular administration (Lanari et al., 2001).
Genotoxic studies: Medroxyprogesterone acetate was reported to
be negative in Ames test (Zeiger et al., 1992; Lang and Reimann,
1993) and V79 cells in vitro (Herzog and Leuschner, 1995). It was
reported positive for unscheduled DNA synthesis in rat hepatocytes in
vivo (Martelli et al., 2003), but negative in cultured human
hepatocytes (Martelli et al., 2003). It does not induced micronucleus
in mouse bone marrow cells (Morita et al., 1997) and chromosomal
aberrations in rat bone marrow cells (Herzog and Leuschner, 1995). It
did not form DNA adducts in human lymphocytes (Werner et al., 1997)
and was reported to form DNA adducts in human liver slices in vitro
(Feser et al., 1998). It induced chromosomal aberrations and sister
chromatid exchanges in cultured human peripheral blood lymphocytes only
in the presence of metabolic activation supplemented with NADP (Siddique
et al., 2006a).
Chlormadinone acetate (CAS No. 302-22-7)
(6-chloro-17-hydroxypregna-4,6-diene-3, 20-dione acetate)
Colour: White or Creamy white
Solubility: Chlormadinone acetate soluble in water, sparingly
soluble in alcohol, soluble in acetone, ether and dimethysulphoxide.
Postulated mode of action: Chlormadinone acetate (Fig.
5) is a synthetic progesterone analogue. It is a derivative of 17α-hydroxyprogesterone
(pregnanes), having chlorine atom at carbon-6 (Schindler et al.,
2003). Chlormadinone acetate, with its anti-androgenic action (direct
inhibitory effect on prostrate), exerts an inhibitory effect on hypertrophic
prostrate, atrophic effects on prostrate. PROSTAL®-l Tablets contain
50 mg of chlormadinone acetate. When one tablet of PROSTAL-L Tablets was
orally administrated to healthy male adults at the fasting state, the
plasma concentration reached the maximum level at 5.1 h (Tmax)
after the administration with a half life of 10.2 h (T½)
showing the sustained release pattern of plasma concentration compared
with ordinary chlormadinone acetate tablets. After oral intake chlormadinone
acetate is rapidly absorbed and undergoes nearly no first pass metabolism.
Therefore, the bioavailability is nearly 100%. It has no binding affinity
to Estrogen Receptor (ER), Mineralocorticoid Receptor (MR) sex-hormone
binding receptor and Corticosteroid-Binding Globulin (CBG). Chlormadinone
acetate elimination occurs slowly. After 7 days only 34% of the dose is
extracted. The reduction of the 3-keto-group results in the inactivation.
3-hydroxy-chlormadinone acetate is the important metabolite, which shows
70% of the anti-androgenic activity. Hydroxylation occurs at positions
C2α, C3β and C15β. The majority of the metabolites are
excreted renally, predominantly as glucoronides (Schindler et al.,
2003). In cattle Chlormadinone is used for oestrus synchronizations at
daily oral doses of 12 mg per animal for upto 20 days. The minimal effective
level in human was 50 μg day-1 (CVMP, 200). Chlormadinone
acetate is also used in sheep and goats for the same indication at daily
oral doses of 2.5 mg per animal and in horses at daily oral doses of 12
mg per animal for upto 20 days. Its oral LD50 is 6400 mg kg-1
b.wt. but intraperitoneal LD50 in mice is 90 mg kg-1 b.wt.
(Siddique and Afzal, 2004a).
|| Chlormadinone acetate
Cancer studies: Chlormadinone produced mammary tumors in dogs
and also increase the incidence of mammary gland hyperplasia and mammary
nodules (El Etreby and Graf, 1979). There is no data available on the
genetic and related effects of chlormadinone acetate alone in humans.
Genotoxic studies: It was found negative in bacterial test system,
unscheduled DNA synthesis (UDS) in rat hepatocytes in vitro (Topinka
et al., 1995) and chromosomal aberrations in human peripheral blood
lymphocytes in vitro (Stenchever et al., 1969), but in other
study was reported to induced chromosomal aberrations and sister chromatid
exchanges in cultured human lymphocytes (Siddique and Afzal, 2004b, 2005a;
Siddique et al., 2008a). It was reported to form DNA adducts in
rat liver in vitro (Topinka et al., 1995; Feser et
al., 1996; Brambilla and Martelli, 2002) and human hepatocytes in
vitro (Topinka et al., 1995; Werner et al., 1997) and
micronucleus in rat liver cells in vivo (Martelli et al.,
Ethynodiol diacetate CAS No. (297-76-7)
Colour: White or almost white
Odour: Odourless or almost odourless
Solubility: It is practically insoluble in water, soluble in alcohol,
chloroform, ether and dimethyl sulphoxide. It is sparingly soluble in
Postulated mode of action: Ethynodiol diacetate (Fig.
6) is a derivative of 19-nortestosterone (estranes). It is used in
the treatment of hypermenorrhea (menorrhagia), pain associated with endometriosis,
dysmenorrhoea and dysfunctional uterine bleeding. It is also used as a
female contraceptive. Ethynodioldiacetate tricks the body processes into
thinking that ovulation has already occurred, by maintaining high levels
of the synthetic progesterone. This prevents the release of eggs from
ovaries. Ethynodiol diacetate and ethinylestradiol combination is used
in oral contraceptives i.e., Demulen® and Zovia®.
This combination prevents pregnancy by preventing ovulation (egg release).
It causes a variety of hormonal changes. To prevent pregnancy after intercourse,
the medicine either prevents or delays ovulation (egg release). Ethynodiol
diacetate binds to the progesterone and estrogen receptors target cells
include the female reproductive tract, mammary gland, the hypothalamus
and the pituitary. Once bound the receptor, it shows the reduction in
the frequency of release of Gonadotropin Releasing Hormone (GnRH) from
the hypothalamus and blunt preovlatory LH (Lutenizing hormone) Surge.
It is metabolized in liver or gut wall, to norethisterone and to sulphate
and glucoronide conjugates. Its half life is about 25 h.
|| Ethynodiol diacetate
Cancer studies: Ethynodioldiacetate increased the incidence of
benign liver tumours in male mice and of mammary tumours in castrated
male mice. There is no data available on the genetic and related effects
of ethynodiol diacetate alone in humans. Oral administration of ethynodiol
diacetate; in combination with mestranol to mice, increased the incidence
of pituitary tumours. Ethynodiol diacetate plus ethinylestradiol increased
the incidence of pituitary tumours and of malignant tumours of connective
tissues of the uterus in mice. In rats it produced mammary tumours.
Genotoxic studies: Ethynodiol diacetate was reported to induced
chromosomal aberrations and sister chromatid exchanges on cultured human
lymphocyte in the presence of metabolic activation supplemented with NADP
(Siddique and Afzal, 2004c; Siddique et al., 2007a).
Megestrol acetate (CAS: 595-33-5)
Colour: White or creamy white
Odour: Odourless or almost odourless
Solubility: Megestrol acetate is practically in soluble in water,
sparingly soluble in alcohol, very soluble in chloroform, slightly soluble
in ether and in fixed oils.
Postulated mode of action: Megestrol acetate (Fig.
7) is a derivative of 17α-hydroxy progesterone (Pregnanes). It
is used in oral contraceptives, breast and in the treatment of endometrial
cancer. After oral intake the bioavailability of megestrol acetate is
100%. It is not bound to Sex Hormone Binding Protein (SHBG) or Corticosteroid
Binding Globulin (CBG). It is bound to serum albumin. The most important
metabolic pathways are hydroxylation reactions. It is metabolized by liver
to glucoronide conjugates. It is excreted as conjugates metabolites via
the urine and faeces (Schindler et al., 2003).
|| Megestrol acetate
Cancer studies: Megestrol acetate plus ethinylestradiol has also
been tested for carcinogenicity by oral administration in mice and rats.
In mice, an increased incidence of malignant mammary tumours was observed
in both sexes, but no increase in incidence of tumours was seen in rats.
There is no data available on the genetic and related effects of megesterol
acetae alone in humans (Joosten et al., 2004).
Genotoxic studies: It has been reported negative in unscheduled
DNA synthesis test using rat hepatocytes; however, the presence of DNA
adducts has been shown in rat liver in vivo and cultured human
hepatocytes (Topinka et al., 1995; Feser et al., 1996; Werner
et al., 1997). It has also been shown to induce micronucleus in
rat liver in vivo, but has failed to cause chromosomal aberrations
in human peripheral blood lymphocytes in vitro (Stenchever et
al., 1969; Martelli et al., 1996). It induced chromosomal aberrations
and sister chromatid exchanges in mice bone marrow cells (Siddique et
Cyproterone acetate (CAS No: 427-51-0)
Colour: White or creamy white
Odour: Odourless or almost odourless
Solubility: Cyproterone acetate is practically insoluble in water,
sparingly soluble in alcohol, very soluble in chloroform, acetone and
dimethysulphoxide acetone, slightly soluble in ether and in fixed oils.
Postulated mode of action: Cyproterone acetate (Fig.
8) is a derivative of 17α-Hydroxyprogesterone (Pregnanes). In
addition to the 6, 7 double bond, the 1,2 α-methyl group is present.
Cyproterone acetate is a potent steroidal antiandrogen with progestational
activity. It is used alone or in combination with ethinylestradiol or
estradiol valerate in the treatment of women suffering from disorders
associated with androgenization, e.g., acne or hisuitism. Cyproterone
acetate competes with dihydrotestosterone for the androgen receptor and
inhibits translocation of the hormone receptor complex in to the cell
nucleus (Sciarra et al., 1990). The bioavailablity is nearly 100%.
Cyproterone acetate has no binding affinity to sex hormone binding globulin
and corticosteroid binding globulin in the serum but 93% of compound is
bound to serum albumin. It is stored in fat tissue and excreted slowly.
The important metabolic steps are hydroxylation reaction and de-acetylation.
The metabolite 15β hydroxycyproterone acetate shows only 10% of the
progestogenic potency of cyproterone itself. The bio-activation of the
cyproterone acetate involves the reduction of the keto group at carbon-3,
which is followed by sulfonation of the hydroxy steroid. The resulting
sulfoconjugate is supposed to be very unstable and can decompose to a
reactive DNA binding carbonium ion (Schindler et al., 2003).
|| Cyproterone acetate
Cancer studies: Cyproterone acetate is a tumour initiating agent
in the liver of female rats (Deml et al., 1993).
Genotoxic studies: Cyproterone acetate was found negative in V79
cells in vitro (Lang and Reimann, 1993; Kasper et al., 1995)
in Ames test (Lang and Reimann, 1993) and for micronucleus in mouse bone
marrow cells in vivo (Reimann et al., 1996; Martelli et
al., 1996) but it was positive for micronucleus in rat liver in
vivo, for chromosomal aberrations in V79 cells (Kasper et al.,
1995) and the human peripheral blood lymphocytes in vitro (Reimann
et al., 1996). It induces mutation in Big Blue rat (Krebs et
al., 1998). It was also found positive for unscheduled DNA synthesis
(UDS) test in rat hepatocytes (Neumann et al., 1992; Kasper et
al., 1995; Topinka et al., 1995; Martelli et al.,
1995), human hepatocytes in vitro and in rat liver in vivo
(Kasper and Muller, 1996) In female rats, DNA adducts have been observed
at low doses of cyproterone acetate, which are in the range of therapeutic
doses used in women (Werner et al., 1997). It was found to induce
chromosomal aberrations and sister chromatid exchanges in human lymphocytes
in vitro (Siddique and Afzal, 2005b, 2004d; Siddique et al.,
2006b, 2007b, 2008b).
Lynestrenol (CAS No 52-76-6) (17α)-19-Norpregn-4-en-20yn-17ol)
Solubility: Lynestrenol is practically insoluble in water, sparingly
soluble in alcohol, very soluble inacetone, chloroform, dimethysulphoxide.
Postulated mode of action: Lynestrenol (Fig. 9)
is used as single cavity drug or in combination with estrogen, such as
ethinylestradiol or mestranol in oral contraceptives (Siddique and Afzal,
2004e). It is the derivative of 19-nortestosterone. Lynestrenol is converted
in vivo to norethisterone. It is metabolized by 3β-hydroxylation
and dehydrogenation (Schindler et al., 2003).
Cancer studies: The high doses of lynestrenol were associated
with the increases incidence of mammary nodules and carcinomas (Misdorp,
1991). The LD50 value for lynestrenol for mice is 110 mg kg-1
body weight (Siddique and Afzal, 2005c).
Genotoxic studies: Lynestrenol induced chromosomal aberrations
and sister chromatid ecology at the dosages of 13.75 and 27.50 mg kg-1
body weight in mice bone marrow cells (Siddique and Afzal, 2005c). It
induced CA and SCE in cultured lymphocyte only in presence of metabolic
activation (Siddique and Afzal, 2004e).
Norgestrel (CAS : 797-63-7)
Colour: White or almost white
Solubility: Norgestrel is practically insoluble in water, sparingly
soluble in alcohol, methylene chloride and freely soluble in chloroform
and dimethyl sulphoxide.
Postulated mode of action: Norgestrel (Fig. 10)
is the derivative of 19-nortestosterone (gonanes). It is a combination
of both active and inactive enantiomers of which only the levorotary form
is biologically active. It is used as a oral contraceptive either as single
agent or in combination with an estrogen. Norgestrel was approved by the
FDA in 1973. Norgestrel after oral administration is readily absorbed
from the gastrointestinal tract and is widely distributed in body fluids.
Protein binding is > 90% and is primarily to Sex Steroid Binding Globulin
(SSBG) and albumin. Its half life is 10.26 h. Metabolism is believed to
be hepatic with an elimination half life of about 20 h. Elimination is
mostly via the urine with minimal amounts excreted in the bile and milk.
Approximately 0.1% of the daily dose passes into breast milk (Schindler
et al., 2003).
Cancer studies: It is reported to increase endometrial thickness
during intrauterine progesterone therapy (Kresowik et al., 2008).
Genotoxic studies: It was reported to be genotoxic in cultured
human peripheral blood lymphocyte with and without metabolic activation
(Ahmad et al., 2001) and was negative for micronucleus in mouse
bone marrow cells (Jordan, 2002). Norgestrel induced chromosomal aberration
and sister chromatid exchanges in the presence of metabolic activation
supplemented with NADP in culture human lymphocytes (Siddique et al.,
Norethisterone (CAS No : 68-22-4)
Colour: White or yellowish white
Solubility: Norethisterone is insoluble in water, sparingly soluble
in alcohol and soluble in chloroform and dimethylsulphoxide.
Postulated mode of action: Norethisterone (Fig.
11) is also named norethindrone and is often used as norethisterone
acetate (NETA). Both compounds are rapidly absorbed from the gastro intestinal
tract. The bio availability is about 64; 36% are bound to sex hormone
binding globulin; 61% to serum albumin and 3% are free in circulation.
Its half life is 5-12 h and is metabolised in the intestinal wall and
liver. The principal metabolite is 5-α-dihydronorethisterone (Schindler
et al., 2003).
Cancer studies: Norethisterone and its acetate were tested
by oral administration in mice and rats and by subcutaneous implantation
in mice. In mice, norethisterone and its acetate increased the incidence
of benign liver cell tumours in males. It increased the incidence of pituitary
tumours in females and produced granulosa cell tumours in the ovaries
of females. It also increased the incidence of benign liver cell tumours
and benign and malignant mammary tumours in male rats (Ahmad et al.,
Genotoxic studies: Northisterone has been found to be negative
in bacterial tests (Lang and Reimann, 1993; Dhillon and Dhillon, 1996).
The widely used contraceptive steroid has been studied with respect to
SCE and UDS induction and DNA adducts formation, in a range of in vitro
and in vivo systems. Induction of UDS in human and rat hepatocytes
was either positive or variable (Martelli et al., 2003). A metabolic
gender difference was found : male but not female hepatocytes gave UDS
response (Joosten et al., 2004), 32-P-post labelling was negative
in rat liver in vivo (Feser et al., 1996) SCE induction
in human lymphocytes was negative in one study (Ahmad et al., 2001),
but positive in another study (Dhillon and Dhillon, 1996). Administration
of norethisterone to mice caused SCE formation in bone marrow cells (Dhillon
and Dhillon, 1996). Positive, negative and variable results have been
obtained in tests for chromosomal aberrations or micronucleus induction
with norethisterone (Dhillon and Dhillon, 1996; Stenchever et al.,
1969; Martelli et al., 1998).
Norethynodrel (CAS No : 68-23-5)
(17 α)-17-hydroxyl-19-norpregn-5 (10) en-20yn-3-one)
Colour: White or almost white
Solubility: Practically insoluble in water; soluble in alcohol,
Cancer studies: Norethynodrel (Fig. 12) was
tested by oral administration in mice and rats and by subcutaneous implantation
in mice. It increased the incidence of pituitary tumours in mice of each
sex and that of mammary tumours in castrated mates of one strain. It also
increased the incidence of benign and malignant liver cell, pituitary
and mammary (benign and malignant) tumours in male rats (Jordan et
Genotoxic studies: Norethynodrel was negative for ames test (Lang
and Reimann, 1993). For unscheduled DNA synthesis it was positive in male
rat hepatocytes in vitro, but negative in rat hepatocytes in
vitro (Joosten et al., 2004). Norethynoderel did not induced
aneuploidy in human cells in culture or unscheduled DNA synthesis in rat
hepatocytes in vitro it inhibited inter cellular communications
in Chinese hamster V79cells (Joosten et al., 2004). It induced
chromosomal aberrations and sister chromatid exchanges in the presence
of metabolic activation supplemented with NADP in cultured human lymphocytes
(Siddique and Afzal, 2005d; Siddique et al., 2007d, 2006d) and
chromosomal aberrations and sister chromatid exchanges at 15.62 and 31.25
mg g-1 body weight in mice bone marrow cells with a LD 50 dose
of 125 mg kg-1 (Siddique and Afzal, 2003).
Dimethisterone (CAS No : 79-64-1)
Solubility: It is insoluble in water, freely soluble in dehydrated
alcohol, very soluble in chloroform, acetone, dimethyl sulphoxide.
Cancer studies: Dimethisterone (Fig. 13) was
tested for its carcinogenicity in various animal models. There is no increase
in tumour incidence (Drill, 1980).
Genotoxic studies: It did not induced chromosomal aberration in
cultured human peripheral blood lymphocytes (Stenchever et al.,
Progesterone (CAS No : 57-83-0)
(Preg-4-ene-3, 20 dione)
Colour: White or slightly yellowish white
Solubility: It is practically insoluble in water, freely soluble
in dehydrated alcohol, very soluble in chloroform, sparingly soluble in
acetone, in ether, in fixed oils and dimethysulphoxide.
Postulated mode of action: Progesterone (Fig. 14)
is used in oral contraceptives and in cure of premenstrual syndrome. It
is 95-98% bound to plasma proteins and is metabolized as glucoronide conjugate
by the liver (Schindler et al., 2003).
Cancer studies: Progesterone was tested by subcutaneous and intra
by intramuscular injection in mice, rabbits and dogs and by subcutaneous
implantation in mice. It increased the incidences of ovarian, uterine
and mammary tumours in mice. Neonatal treatment with progesterone enhanced
the occurrence of pre-cancerous and cancerous lesions of the genital tract
and increased mammary tumorigenesis in female mice (Lamb et al.,
Genotoxic studies: Progesterone did not induce dominant lethal mutations
in mice or chromosomal aberrations in rats treated in vivo. It
did not induced chromosomal aberration or sister chromatid exchange in
cultured human cells, nor chromosomal aberrations or DNA strand breaks
in rodent cells. Studies on transformation of rodent cells in vivo
were inconclusive; a clearly positive result was obtained for rat embryo
cells, are weakly positive result for mouse cells and a negative result
for Syrian Hamster embryo cells. Progesterone was not mutagenic to bacteria
(Joosten et al., 2004) The test results of the L5178-YTK mouse
lymphoma assay were inconclusive (Myhr and Caspary, 1988), All data a
UDS induction in male rat or human hepatocytes (Oshiro et al.,
1986; Martelli et al., 2003) or 32P-post labelling in
the rat liver in vivo or human hepatocytes (Feser et al.,
1996; Werner et al., 1997), indicate that progesterone does not
cause DNA lesions variable responses with inter-animal differences in
UDS were observed in male and female rat hepatocytes (Martelli et al.,
2003). Progesterone was not clastogenic in cultured human lymphocytes
(Stenchever et al., 1969).
Increased frequencies of micronuclei were found in hepatocytes of rats
treated with a single oral dose (100 mg kg-1) of progesterone
(Martelli et al., 1998). It does not induce an euploidy in DON
cells in vitro (Wheeler et al., 1986).
MECHANISM FOR THE GENTOTOXICITY
The carcinogenic effects of hormone replacement therapy used to relieve
symptoms of menopause were evaluated by the International Agency for Research
on Cancer (Joosten et al., 2004). Most of the studies reviewed
did not differentiate between the effects of estrogen only and estrogen-progestin
combination therapies. An increased risk of endometrial cancer was associated
with increasing duration of therapy (Kresowik et al., 2008). Numerous
case control and cohort studies have addressed the risk of various cancers
associated with the use of oral contraceptives (Heinemann et al.,
1997). Most of the studies involved estrogen-progestins combinations.
Studies in rats, mice, hamsters and guinea pigs have been conducted with
estrogens alone or in combination with known carcinogens. Estrogen had
a carcinogenic effect in all species and by all routes of administration.
Most studies showed induction of benign and malignant neoplasias as well
as preneoplastic lesions, in a variety of target organs, including the
breast and female reproductive tract (Joosten et al., 2004).
Steroidal estrogens can damage chromosomes and DNA in mammals (Banerjee
et al., 1994; Siddique and Afzal, 2004f; Siddique et al.,
2005b). The most frequently reported effects include DNA adduct formation,
cytogenetic alterations (e.g chromosome and chromatid breaks, micronuclei,
SCEs), aneuploidy and cell transformation (Djelic and Djelic, 2002). The
genotoxic effects of estrogens/synthetic progestins/androgens have been
demonstrated in various in vitro assays, using cultured animal
cells or cell free systems. Fewer effects have been reported in whole
animal studies or in studies with human cells and no human in vivo
studies were identified (Siddique et al., 2007e; Beg et al.,
2007; Joosten et al., 2004; Djelic et al., 2005).
Many different formulations of synthetic and naturally produced estrogens
are prescribed for use as oral contraceptives or in postmenopausal hormone
replacement therapy (Biri et al., 2002). Exogenous estrogens are
well absorbed from the gastro intestinal tract and the skin of human animals.
Estrogens are metabolized in the gastro intestinal and other tissues.
In both human and animals, estrogens undergo similar phase I and Phase
II reactions. Aromatic hydroxylation reactions catalyzed by cytochrome
P-450 enzymes are the primary phase 1 pathways. (Chen et al., 1998).
Sulfation, methylation and glutathione conjugation are the major phase
2 pathways. The major phase 1 metabolic pathway for endogenous estrogens
is aromatic hydroxylation to catechol intermediates. Concerning the genotoxic
effects of estrogens; 16α-Hydroxyestrone, 4-hdroxyestradiol and 4-hydroxyestrone
have direct genotoxic effects and carcinogenicity. Liehr et al.
(1986) described mechanistic similarities between human breast cancer
and estrogen induced kidney cancer in hamsters and identified metabolism
to the 4-hydroxylated catechols as the primary pathway leading to tumour
development. The 4-hydroxylated catechols may undergo subsequent redox
cycling between semiquinone and quinone forms. The quinones may undergo
nonenzymatic isomerization to quinone metabolites. The quinone and quinone
metabolites intermediates are highly reactive and may form covalent DNA
adducts, thus these metabolites are candidates for ultimate estrogen carcinogens.
Redox cycling between various forms of quinones generates superoxide radicals
that are capable of direct and indirect damage to DNA. In breast cancer
cells, 4-hydroxylation predominates over 2-hydroxyltion (Zhu and Conney,
Excessive production of reactive oxygen species has been reported in
breast cancer tissue and free radical toxicity (DNA single strand breaks,
lipid peroxidation and chromosomal abnormality) has been reported in hamsters
treated with estradiol. Reactive oxygen species, including superoxide,
hydrogen peroxide and hydroxyl radicals, may be produced through redox
cycling between the O-quinones and their semi quinone radicals (Roy et
al., 1991). These reactive oxygen species can cause oxidative cleavage
of the phosphate-sugar back bone and oxidation of the purine and pyrimidine
residues of DNA. The incubation of 4-hydroxylated catechols with microsomes,
NADPH and DNA resulted in 8-hydroxylation of guanine bases. 8-hydroxy-deoxyguanosine
is a biomarker for oxidative damage and is considered an important factor
in carcinogensis (Han and Liehr, 1994; Bolton et al., 1998; Bolton,
2002). The possible mechanism and cause of the genotoxicity at different
dosages of synthetic progestins such as cyproterone acetate, Chlormadinone
acetate, medroxy progesterone acetate, have been studied by using different
doses of superoxide dismutase and catalase in the presence of metabolic
activation with and without NADP. Superoxide dismutases (SODs) are family
of metal enzymes that convert O.-2 to H2O2
according to the following reaction (Culotta, 2000).
Catalase is a heme containing protein that brings about the decomposition
of H2O2 into water and oxygen. Catalase is found
to reduce sister chromatid exchanges levels. The mechanism of reaction
is (Reid, 2003):
The treatment of SOD with ethinylestradiol in the presence of metabolic
activation with NADP has been reported to increase the genotoxic damage
(Siddique et al., 2005b). Since ethinyl estradiol is genotoxic
only in the presence of metabolic activation with NADP, the first step
would involve the aromatic hydroxylation catalysed by cytochrome P450,
as occurs in the case of estrone and the 17β-estradiol forming catechol
metabolites (Yager and Liehr, 1996; Bolton et al., 1998). Cytochrome
P450, in liver S9 fractions plays an important role in activating promutagens
to proximated/or ultimate mutagens. Rat and human liver P450 is involved
in the activation of some chemical carcinogens having different isoforms
(Maron and Ames, 1983; Guengerich and Shimada, 1991). In the tissues of
liver, 2-hydroxylation predominates over 4-hydroxylation by approximately
9:1, however, in extra hepatic tissues the ratio drops to 1:1 (Zhu and
Conney, 1998). Only 4-hydroxyestrone-3, 4-dihydroxy 1,3,5 (10)-oestraien-17one
(4-OHE) was found to be carcinogenic in the male Syrian golden hamster
kidney tumour model, whereas 2-OHE was found to be without any activity
(Yager and Liehr, 1996; Guengerich and Shimada, 1991). Once formed, the
endogenous catechol estrogens can be oxidized by virtually any oxidative
enzymes in the absence or presence of metal ion and can give rise to o-quinones
(Yager and Liehr, 1996; Bolton et al., 1998). The study on ethinylestradiol
confirms the presence of Reactive Oxygen Species (ROS) and the source
of reactive oxygen species has been suggested to be the result of redox
cycling between o-quinones and their semiquinone radicals, generating
superoxide, hydrogen peroxide and ultimately reactive hydroxyl radicals,
that cause oxidative cleavage of the phosphate sugar backbone as well
as oxidation of the purine/pyrimidine residues of DNA (Han and Liehr,
||Possible mechanism of generating free radicals by megestrol
acetate (MGA) (Siddique et al., 2005a; Hum Exp Toxicol)
The study on cyproterone acetate, chlormadinone acetate and megestrol
acetate reveal that the compounds increases chromosomal aberrations and
frequencies of sister chromatid exchange, significantly in cultured human
lymphocytes (Siddique and Afzal, 2004b, 2005a, b). The treatment with
superoxide dismutase, increased the frequencies of chromosomal aberrations
and sister chromatid exchanges generated by both cyproterone acetate and
chlormadinone acetate. The possible mechanism of free radical generation
for cyproterone acetate, megestrol acetate and chlormadinone acetate is
shown in Fig. 15-17.
XOOH can also give rise XOO• (alkoxyl) and •OOH
(Peroxyl) radicals. The presence of alkoxyl (XOO•) radical
appears to be a very remote possibility in view of the highly polar nature
of the living system, because X would have to be essentially an alkyl
group (Islam et al., 1991). Hydroxyl radical (OH) is converted
into hydrogen peroxide by superoxide dismutase (Culotta, 2000), as a result,
the increase in chromosomal aberrations and sister chromatid exchanges
was observed when the cyproterone acetate and chlormadinone acetate were
treated with superoxide dismutase. The catalase treatment with cyproterone
acetate and chlormadinone acetate have been reported reduce the genotoxic
damage (Siddique and Afzal, 2004b, 2005a, b).
Megestrol acetate has been reported to be genotoxic in mice bone marrow
cells (Siddique et al., 2005a). The treatment of megestrol acetate
with ascorbic acid reduced the genotoxic damage of megestrol acetate.
Ascorbic acid posses a substantial nucleophilic character and it has been
suggested that ascorbate might protect against electrophilic attack a
cellular DNA by intercepting reactive agents (Edgar, 1974). Reactive oxygen
species generated by MGA via nucleophilic reaction have been suggested
responsible for the genotoxic damage (Siddique et al., 2005a).
Norgestrel shows genotoxic effects only in the presence of metabolic activation
in the presence of NADP (Siddique et al., 2006c). The metabolic
activation of norgestrel and possible conversion of it to reactive species
may be responsible for its genotoxicity. Metabolic activation of estrogens
such as estradiol-17β and ethinyl estradiol results in the production
of reactive oxygen species via redox cycling between quinones and semi-quinone
radicals (Fig. 18) (Siddique et al., 2005b).
||Possible mechanism of generating free radicals by cyproterone
acetate (Siddique and Afzal, 2005b; Toxicol in vitro)
||Possible mechanism of generating reactive oxygen species
by chlormadinone acetate (Siddique and Afzal, 2004b) Indian J Exp
Medroxyprogesterone acetate (Fig. 19) showed genotoxic
effects in the presence of metabolic activation supplemented with NADP
(Siddique et al., 2006a).The genotoxic potential of various synthetic
progesting has been tested in different experimental models using different
genotoxic end points (Joosten et al., 2004). Very little attention
has been paid to the structural relationship and the potentiality to induce
the genotoxic damage by synthetic progestins. Structural relationship
and ability to induce genotoxic damage has been worked out, using DNA
repair assay and micronucleus formation in the liver of female rats (Brambilla
and Martelli, 1997). For DNA repair following trend was found, viz. cyproterone
acetate > chlormadinone acetate > megestrol acetate. It was reported
that cyproterone acetate with (a) a1, 2α-methylene group, (b) a keto
group at carbon-3, (c) two double bonds, C4 = C5
and C6 = C7 and (d) Cl at carbon-6, is the most
genotoxic molecule. The lower genotoxic potencies of chlormadinone acetate
and megestrol acetate might well be due to the absence of 1, 2α-methylene
group and for megestrol acetate, perhaps due to the presence of methyl
group (-CH3) at carbon-6 instead of Cl (Brambilla and Martelli,
1997). However, when the adduct formation in cyproterone acetate, medroxyprogesterone
acetate, megestrol acetate and chlormadinone acetate are considered, the
cyproterone acetate, megestrol acetate and chlormadinone acetate are found
to form adducts in human liver slices in vitro. Medroxyprogesterone
acetate forms these at low level (Feser et al., 1998).
||Possible metabolism ethinylestradiol to catechols and
quinones and generation of reactive oxygen species by redox cycling
of 4-hydroxy equilenin (Siddique et al., 2005b, Chem Biol Interact)
||Possible metabolism and generation of reactive oxygen
species by medroxyprogestrone acetate (Siddique et al., 2006a
Studies performed on megestrol acetate (Siddique et al., 2005a),
cyproterone acetate (Siddique and Afzal, 2005b), chlormadinone acetate
(Siddique and Afzal, 2004b), ethynodiol diacetate (Siddique and Afzal,
2004c), norethynodrel (Siddique and Afzal, 2005d), norgestrel (Siddique
et al., 2006c) and lynestrenol (Siddique and Afzal, 2004e), show
that the progestins, in which double bond between carbon-6 and carbon-7
is present, like megestrol acetate, cyproterone acetate and chlormadinone
acetate, can undergo nucleophilic reaction and generate free radicals
in the system and therefore, show the genotoxic effects and the progestins,
in which double bond between carbon-6 and carbon-7 is absent, may require
additional metabolic activation to show the genotoxic effects in the test
system. Looking at the chemical structure of estradiol and ethinylestradiol,
the double bond is found to be absent between carbon-6 and carbon-7. It
is well known that estradiol undergoes aromatic hydroxylation in liver
by cytochrome P450s and results in the formation of catechol metabolites
(Maclusky et al., 1981; Fishman, 1983; Liehr et al., 1986;
Li and Li, 1987; Bolton, 2002). The redox cycling between O-quinones and
their semi-quinone radicals results in the generation of superoxide, hydrogen
peroxide and ultimately reactive hydroxyl radicals which cause oxidative
cleavage of the phosphate-sugar backbone as well as oxidation of the purine/pyrimidine
residues of DNA (Han and Liehr, 1994). Ethinylestradiol generates reactive
oxygen species in the presence of metabolic activation with NADP (Siddique
et al., 2005b). The study performed on norgestrel, ethynodioldiacetate,
lynestrenol and norethynodrel shows that they exhibit genotoxic effects
only in the presence of metabolic activation with NADP. The similarity
in the structure of the above synthetic progestins and the estrogens lies
mainly in the absence of double bond between carbon-6 and carbon-7 (Fig.
Moreover, if the adduct formation on the basis of presence and absence
of double bond between carbon-6 and carbon-7 is considered, a definite
correlation is established. A study, carried out by Feser et al.
(1998) on some selected sex-steroids, shows that chlormadinone acetate
(C6 = C7) and megestrol acetate (C6 =
C7) from DNA adducts, while norgestrel (C6-C7),
estradiol (C6-C7) and ethinylestradiol (C6-C7)
and ethinylestradiol (C6-C7) do not form DNA adducts
in human liver slices. it is thus concluded that the double bond between
carbon-6 and carbon-7 plays an important role in determining the mode
of the genotoxicity of synthetic progestins. On the basis of above information
the model for the genotoxicity of steroids (synthetic progestins/estrogens)
is suggested as shown in the Fig. 21. Estrogens have
been reported to induce genotoxicity in various in vitro and in
vivo models. Synthetic progestins are at second position. Estrogens
caused genotoxicity by metabolic activation i.e., forming catechol estrogens.
The balance of toxification and detoxification determines the responsiviness
by an individual and it is species and tissue dependent. The natural estrogen
can cause DNA damage in the following ways (Joosten et al., 2004).
||Structure of synthetic progestins. Presence of double
bond between carbon-6 and carbon-7 (A to C. Absence of double bond
between carbon-6 and carbon-7 (D-I). (Siddique et al., 2007c;
||A model for the genotoxicity of synthetic progestins
||Redox cycling of catechol estrogen gives rise to reactive
oxygen species (O2-) that can cause single strand
||Hydroxyl radical can oxidized DNA base (Guanine)
||Lipid peroxidation may also take place and results in the formation
of malonaldehyde DNA adducts
||Estrogen-DNA adducts may be formed after metabolic activation
For the synthetic progestin present study shows that the presence of
double bond between carbon-6 and carbon-7 might be important for the genotoxicity.
The presence of double bond is and the adduct formation is also correlated
with the study performed on few selected synthetic progestins by Fesar
et al. (1998). The contradictory results for the studied synthetic
progestins may be due to the low robustness of the various test models
(Clastogenicity). In vitro conditions may generally favour phase-I
activation reactions while not allowing phase-II de-activation reactions.
The metabolic activation is a necessary step for the genotoxicity of natural
estrogens as well as for the synthetic progestins. Some synthetic progestins
are also genotoxic in the absence of metabolic activation (Joosten et
al., 2004). The dose is an important factor in determining the genotoxicity
testing. Dose response relationship must be studied to reach any conclusion.
Generally high doses of estrogens and synthetic progestins have been studied
in various in vitro experimental models i.e., in μM and μg.
The therapeutic plasma concentrations are often in the nanogram or pictogram
mL-1 range. The high doses studied are significant because
this dose range may reach in some clinical abnormal conditions or due
to lack of metabolizing enzymes or other factors (Martelli et al.,
2003). As it is evident from the epidemiological studies that prolonged
users of steroids, are at risk of having various types of cancers. The
dose factor and the clastogeny as its own importance. An increase in the
frequency of chromosomal aberrations in peripheral blood lymhocytes is
associated with an increase in overall risk of cancer (Hagmar et al.,
The steroids are genotoxic only at high doses. The therapeutic doses
are safe. The care should be taken with regard to their concentration
as they may be carcinogenic in the long term use in humans.
Thanks are due to the Chairman, Department of Zoology, Aligarh Muslim
University, Aligarh for providing laboratory facilities. The research
grant (No:09/112(0355)/2003-EMR-1) from the Council of Scientific and
Industrial Research, New Delhi and Department of Science and Technology
(SR/FT/LS-003/2007), New Delhi are also acknowledged.
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