Oestrogen receptors

 

 Dynamics and Vagaries of oestrogen signalling in males and females.!!!  ERs (Oestrogen receptors) have five domains with distinct functions .

 

Q.1: Which site of oestrogen receptor is most dynamic and not just lazy like Dr. Pal?? The N-terminal of the A/B domains of ERs consist of activation function 1 (AF1), which contributes to the transcriptional activity of ERs and is an essential domain for interaction with co-regulators.

Q. 2: How many of us believe that Oestrogens exerts its action via nuclear receptors(NRs-Nuclear Receptors)  unlike insulin?? . Ans:              Insulin receptors act on cytoplasmic mm but estrogen receptors (ERs) which belong to the steroid hormone superfamily of nuclear receptors (NRs) are situated at nuclear mm.

 Q,  3 :What  are the Other types of steroid receptors  exhibiting  action as of nuclear receptors?  Ans: Other types of steroid receptors exerting their actions on nuclear receptors among the NRs(nuclear receptors)  include the estrogen-related receptors (EER), progesterone receptors (PR), androgen receptors (AR), glucocorticoid, and mineral corticoid receptors.

 

Q.4:-Estrogens, a class of steroid hormones, regulate the growth, development, and physiology of the human reproductive system. Estrogens also involve in the neuroendocrine, skeletal, adipogenesis, and cardiovascular systems. Estrogen signaling pathways are selectively stimulated or inhibited depending on a balance between the activities of estrogen receptor (ER) α or ERβ in target organs. ERs belong to the steroid hormone superfamily of nuclear receptors, which act as transcription factors after binding to estrogen. The gene expression regulation by ERs is to modulate biological activities, such as reproductive organ development, bone modeling, cardiovascular system functioning, metabolism, and behavior in both females and males. Understanding of the general physiological roles of ERs has been gained when estrogen levels were ablated by ovariectomy and then replenished by treatment with exogenous estrogen. This technique is not sufficient to fully determine the exact function of estrogen signaling in general processes in living tissues. However, a transgenic mouse model has been useful to study gene-specific functions. ERα and ERβ have different biological functions, and knockout and transgenic animal models have distinct phenotypes. Analysis of ERα and ERβ function using knockout mouse models has identified the roles of estrogen signaling in general physiologic processes. Although transgenic mouse models do not always produce consistent results, they are the useful for studying the functions of these genes under specific pathological conditions.

Keywords: Estrogen, estrogen receptors, knockout mice

Estrogen receptors (ERs) belong to the steroid hormone superfamily of nuclear receptors (NRs) .Other types of steroid receptors among the NRs include the estrogen-related receptors (EER), progesterone receptors (PR), androgen receptors (AR), glucocorticoid, and Estrogen also affects the neuroendocrine, skeletal, adipogenic, and cardiovascular systems .

Q.5 : How many oest receptors are there?? The biological functions of estrogen are mediated by binding to the ERs: estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ).

 

Q. 6 : How estrogen signalling is governed ?  Who finally dictate the mode of action in a particular cell ?

Ans: Estrogen signaling is selectively stimulated or inhibited depending upon a balance between ERα and ERβ activities in target organs. ERα has been cloned from human breast cancer MCF-7 cells in 1986 and ERβ was cloned from rat prostate in 1996

Q. 7: Where are gens for oestrogen domiciled  in which chromose ?? ERα and ERβ are encoded by distinct genes located on different chromosomes. The human ERα gene is located on chromosome 6 while the ERβ gene is on chromosome 14 .

Q.8: How many amino acides are there in ERα and in Erβ ??Ans:. The full-length human ERα protein has 595 amino acids and a molecular size of 66 kDa while the full-length human ERβ protein has 530 amino acids and a molecular size of 54 kDa. Similar to other NRs(nuclear receptors) , ERs have five domains with distinct functions . The N-terminal of the A/B domains of ERs consist of activation function 1 (AF1), which contributes to the transcriptional activity of ERs and is an essential domain for interaction with co-regulators. AF1 is the least conserved region with only 30% identity between ERα and ERβ. Functional studies have shown that ERβ has low levels of AF1 activity. The A/B domains also contain amino acids that are targets of post-transcriptional modifications including splicing to stimulate AF1 activity .

The C domain encodes a centrally located DNA binding domain (DBD) essential for sequence-specific binging of ERs to DNA and regulating the expression of target genes .

The D domain, a hinge region, includes amino acid sequences that stimulate nuclear localization signaling and facilitate post-translational modification of ERs, resulting in the activation of ER signaling in cells. Finally, the E/F domain, located in the C-terminal region, contains a ligand-binding domain (LBD) that serves as an interaction site with co-regulators and ligand-dependent activation function 2 (AF2).mineral corticoid receptors.

Catecholoestrogens

 Here we go:--High concentrations The physiologic role of this interaction remains uncertain.

Catechol estrogens have been shown to be potent inhibitors of the COMT-mediated

Action 1:-catechol oestrogens cause Inactivation of catecholamines. Since catechol estrogens exhibit high affinities for the estrogen receptor protein, it is assumed that the Action 2:-

majority of their non-DNA reactive activities are mediated in a mechanism similar to that evoked by ovarian estrogens.

Mode of action:-The catechol estrogen A) bound estrogen receptors to recruit coactivators and corepressor proteins, recent evidence clearly suggests the most active catechol estrogens

B)alter gene expression in certain target cells.

C) Catechol estrogens are generated by the actions of genes encoded by CYP1A1 and CYP1A2 (which catalyze 2-hydroxylation of estrogens), and CYP1B1, which is an estrogen 4-hydroxylase..

Although the catechol estrogens are short-lived in vivo and are postulated to have physiological functions as locally generated signaling molecules, they also yield potent genotoxic molecules implicated in carcinogenesis. The 4-hydroxyestrogens can be oxidized to quinone intermediates that react with purine bases of DNA, resulting in depurinating adducts that generate highly mutagenic apurinic sites .

Quinones derived from the 2-hydroxyestrogens produce stable DNA adducts and are presumed to be less genotoxic. Metabolism of catechol estrogens may also generate oxygen-free radicals.

Catechol estrogens are methylated by catechol-O-methyltransferase, resulting in a catecholamine-like substance. The methylated catechol estrogens have antiangiogenic and antitumor activity through inhibition of hypoxia-inducible factor-1α (HIF-1α), a proangiogenic transcription factor. There is evidence that altered production of 2-methoxyestradiol may contribute to the pathogenesis of preeclampsia through its modulation of antiangiogenic factor production by the placenta.

Catecholestrogens, an important group of estrogen metabolites which possess significant endocrine properties of their own, are synthesized in brain, liver and other tissues by estrogen-2 hydroxylase, a cytochrome-P450 dependent monooxygenase. Estrogen-2-hydroxylase activity can be detected in both liver and brain in the fetal rat.

By eight weeks of age, sexual dimorphism is apparent: male rats have higher specific enzyme activities in both liver and brain than female rats.

Androgenic hormones play a crucial role in the expression of enzyme activity in the adult animal. In utero exposure to phenobarbital results in substantial changes in brain and liver estrogen-2-hydroxylase activities during the first three weeks of life.

Potential Safety Problems

COMT inhibitors may change the metabolism of endogenous catechol products, such as catecholamines and catechol estrogens (Männistö and Kaakkola, 1999). Concerning catecholamines no relevant safety problems have been associated with COMT inhibitors (Kaakkola, 2000). The effect of COMT inhibitors on catechol estrogen metabolism is still poorly studied and known in humans. Catechol estrogens may be involved in, e.g., some hormonal-dependent cancer formation (Creveling, 2003; Zhu, 2002). So far, no data are available that COMT inhibitors increase or decrease cancer incidence in PD patients. In a recent study in PD patients treated with entacapone slightly more prostate cancer cases were observed in an active drug group compared with a placebo group (Stocchi et al., 2010). In earlier entacapone studies this phenomenon has not observed. Another theoretical safety issue may be related to levodopa metabolism when both DDC and COMT pathways are effectively blocked (Fig. 2). There is a risk that the amount of dopa quinone metabolite increases. This metabolite is labile and potentially toxic, e.g., to dopaminergic cells (Kostrzewa et al., 2002). This concern is probably more relevant if brain COMT is effectively inhibited.

EFFECTS ON CATECHOLAMINE TURNOVER

As mentioned above, their interference in catecholamine metabolism is one of the most widely discussed features of catecholestrogens in the brain. To estimate the degree of such interference, we measured dopamine, norepinephrine and epinephrine concentrations and turnover rates in response to 2-hydroxyestradiol-17β and 2-hydroxyestrone (2-OHE1) in adult orchidectomized and adult ovariectomized rats.

Intramuscular injections of 2-OHE2 and 2-OHE1 (50 ng) or their solvent were given at 1000 h. Females received injections on two subsequent days. α-Methyl-paratyrosinemethylester (250 mg free base/kg) was injected i p at 1400 h to block synthesis of catecholamines. The animals, 10 each group, were rapidly killed by decapitation at 1400 h or at I5OO h. The brains were removed immediately and frozen on dry ice. Trunk blood was collected for assay of LH and prolactin. Medial preoptic area (MPO) and the anterior part of the hypothalamus (AMBH) were punched from 0.8 to 1.0 mm thick frozen slices with sharpened needles. The brain tissues were then handled as described by Höhn and Wuttke (1978). Turnover rates were calculated as described by Brodie and colleagues (1966).

In the male 2-OHE2 and 2-OHE1 decreased serum LH levels but had no effect on prolactin secretion. In the females only 2-OHE2 was effective in altering serum hormone levels. It decreased the level of LH and increased the level of prolactin. It seems not surprising that 2-OHE1 had no sigExploring the unknown!!! Catechol amines are partly known to us but do we, the practicing gynaecologits are aware anything what is called as Catechol oestrogens??

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  Srimanta Pal High concentrations of catechol estrogens are necessary to cause measurable inhibition, but the physiologic role of this interaction remains uncertain. Catechol estrogens have also been shown to be potent inhibitors of the COMT-mediated inactivation of catecholamines. Since catechol estrogens exhibit high affinities for the estrogen receptor protein, it is assumed that the majority of their non-DNA reactive activities are mediated in a mechanism similar to that evoked by ovarian estrogens. Although there are few studies of the ability of catechol estrogen bound estrogen receptors to recruit coactivators and corepressor proteins, recent evidence clearly suggests the most active catechol estrogens alter gene expression in certain target cells.
  Steroid Hormones and Other Lipid Molecules Involved in Human Reproduction
  Jerome F. StraussIII, Garret A. FitzGerald, in Yen and Jaffe's Reproductive Endocrinology (Eighth Edition), 2019
  Catechol Estrogens
  Catechol estrogens are generated by the actions of genes encoded by CYP1A1 and CYP1A2 (which catalyze 2-hydroxylation of estrogens), and CYP1B1, which is an estrogen 4-hydroxylase..
  Although the catechol estrogens are short-lived in vivo and are postulated to have physiological functions as locally generated signaling molecules, they also yield potent genotoxic molecules implicated in carcinogenesis. The 4-hydroxyestrogens can be oxidized to quinone intermediates that react with purine bases of DNA, resulting in depurinating adducts that generate highly mutagenic apurinic sites .
  Quinones derived from the 2-hydroxyestrogens produce stable DNA adducts and are presumed to be less genotoxic. Metabolism of catechol estrogens may also generate oxygen-free radicals.
  Catechol estrogens are methylated by catechol-O-methyltransferase, resulting in a catecholamine-like substance. The methylated catechol estrogens have antiangiogenic and antitumor activity through inhibition of hypoxia-inducible factor-1α (HIF-1α), a proangiogenic transcription factor. There is evidence that altered production of 2-methoxyestradiol may contribute to the pathogenesis of preeclampsia through its modulation of antiangiogenic factor production by the placenta

nificant effects in females, since other investigators

Designer oestrogens -Pt II

 

Designer estrogens or  SERMS, are two terms currently being used to describe the actions of a medication that acts as an estrogen on one tissue and an anti-estrogen on others in a same woman . The ideal estrogen medication would switch of or off the effects of estrogen at different sites in the woman's body.

Designer estrogen is an in fact an engineered drug that possesses some, but not all, of the actions of estrogen. also known as selective estrogen-receptor modulator (SERM). For example, raloxifene (brand name: Sevista) is classified as a designer estrogen because, like estrogen, it A) prevents bone loss and B)  lowers serum cholesterol; however,C)  it does not stimulate the endometrial lining of the uterus.

. Tamoxifen is the first SERM used by mainly breast cancer patients and has been shown to block the recurrence of breast cancer in the other breast by 40 percent.

But risks of Tamoxifene ??  The risk associated with Tamoxifen is that it may increase risk of developing blood clots and uterine cancer. Hormone-replacement therapy (HRT) is effective in relieving menopausal symptoms and reduces the risk of osteoporotic fracture, coronary heart disease  

and probably also Alzheimer's disease.But  

^., Long-term use of HRT(Combined HRT)  is associated with adverse effects, of which the most serious is an increased risk of breast cancer. 

Other unwanted effects of long-term HRT include venous thromboembolism 

and endometrial cancer. 

·        For many women, monthly vaginal bleeds are a considerable inconvenience and an important cause of non-compliance, although this problem may be obviated by the use of continuous combined preparations.

 

 

Adv of TMX? Ans: Tamoxifen in postmenopausal womenmay be better Than combined HRT

Osteoporosis Drug Might Replace HRT for Disease Prevention-- Evidence is mounting that a class of drugs known as "designer estrogens" may fulfill the broken promises of hormone replacement therapy, but experts warn that it is too soon to know for sure.

Selective estrogen receptor modulators (SERMs) have been proved to be effective in preventing bone loss in older women, and early studies suggest they may also protect against stroke, heart disease, breast cancer, and even Alzheimer's.

The downside? SERMs like the drug Evista (raloxifene), which is widely prescribed for the prevention and treatment of osteoporosis, do nothing to lessen the symptoms associated with menopause, and may even bring on hot flashes and mood swings, especially when given close to the menopausal transition period. But Evista appears to be an increasingly popular choice for women who once took hormones to prevent the diseases of aging.

"Although SERMs already are in clinical use, the preventive and therapeutic potential of this class of drugs has only begun to be unraveled," researcher Evanthia Diamanti-Kandarakis, MD, and colleagues wrote in the Jan. 1 issue of the American Cancer Society publication Cancer.

SERM researcher Elizabeth Barrett-Connor, MD, agrees, but says the definitive study assessing the benefits of Evista(RLX)  is ongoing. The trial involves just over 10,000 women who are at increased risk for heart disease, and findings are expected in 2006.

"Women and their doctors got a big surprise a few months ago with regard to hormone replacement therapy (HRT)," Barrett-Connor tells WebMD. "We have to be very careful at this point not to recommend [SERMs] for unproven indications so they don't get another one."

Barrett-Connor is referring to the large government study, halted early last July, which showed HRT to be associated with an increased risk for breast cancer, heart attack, stroke, and blood clots.

Raloxifen ( Sevista)  has been approved for the prevention and treatment of osteoporosis, but not for other diseases. In a trial involving 7,000 women, Barrett-Connor and colleagues found that the drug is not associated with an increased risk of cardiovascular disease. Although the results suggest the drug can lower "bad" LDL cholesterol, it had no effect on "good" HDL cholesterol. RLX   significantly reduced the risk of stroke in the same study population, and earlier findings showed a dramatic reduction in breast cancer risk.

Designer estrogens Tibolone

 

SERM: SERM are structurally diverse compounds which act a both as a agonists and antagonists in the same woman and  such agents are also called “Designer Oestrogens”:-

Because the SERMs cause agonist effects on oestrogen receptors  in some tissues of women , while depresses or suppresses some other oestrogen receptors in other parts of tissues in a same individual  .

.

 Classification / Evolution of SERM:- A) First SERM is :CC ( 1982/ 1968)

                                  B) Second SERM:-Tamoxifen (still used as an effective agent or preventive measure as barest cancer after surgery –antagonist effect at Breast).

                              C) Third SERM:-Raloxifene.(This structure looks more like tamoxifen).  

Basics of Designer oestrogens? There are 3 types of E2 receptors 1) Alpha α homo-dimer2) β beta homodimer 3) hetero-dimer α & β receptor heterodimers. They by virtue of their structural stereochemical composition they (designer) affect some spenic receptors and either exerts stimulatory action or inhibitory action.

The idea of designers was to find out such designers which when consumed in  vivo will preserve the beneficial effects of oestrogens but will impede the ill effects of oesrogens.

Limitations of designer oestrogens :--They have not been fully successful in their mission till   date.

Q.7: How we can use such designer oestrogens in menopausal women / post menopause??

Ans: There are two phases of Ry for menopausal women. A) Phase I treatment:- it spans from first few months or years when there are acute menopausal symptoms like hot flashes, mood changes, sleep disturbance,  vaginal  dryness . In such acute phase estrogen will be a better choice either COC or Patch.

Phase II of treatment (HRT):-As we know such phase two Tr is aimed at  prevention of some diseases like Osteopenia, Breast Ca . This phase II tr in established menopause is purely prophylaxis as a measure to achieve long term health maintenance. Raloxifene is an important member of this therapy.

How useful is double marker screening along with NT scan ?

 https://www.facebook.com/photo.php?fbid=1759818267511470&set=gm.3488310827927637&type=3&eid=ARBcKbb_8P7TPjb1eKks-kxlkL9N3fLjpzKcz7O3KWVU6lB5U08O9g6gc6X-x6F5JL-pP2CSK0HCby2P&ifg=1

First trimester screening

 CVS/ Amniocentesis for definitive diagnostic (not screening tets but confirmatory tets) :-All women  with positive results of prenatal screening, and then  karyotyping of a fetus (from CVS / Liquor amni) have to be done  with amniocentesis. Using a sensitivity analysis method, it has been determined with high probability that a pathological value of the marker implies the presence of risk. Using a specificity analysis method, it has been determined with high probability that a normal value of the marker implies the absence of significant risk. Using a positive expected value method, it has been determined with high probability that risk is present only if the marker implies so. Using a negative expected value method, it has been determined with high probability that significant risk is absent if the marker implies so.

If the findings of genetic screening evidence of risk of Down’s syndrome in the PRISCA software greater than 1: 250, which is indicated based on prenatal karyotyping.

In a study it was observed that in sample of 340 high-risk findings of the screening, there were 18 (6.1%) results with the pathological values of NT markers.

Pathological PAPP-A values were found in 174 (59.1%) cases.

Free beta-hCG showed extreme values in 168 (57.1%) pregnant women.

 Values of the markers  are being usually  reported in deviation from the median – MoM (multiple of median).

The risk of Down’s syndrome is shown in PRISCA software at two levels, the risk of biochemical correlations of biochemical markers PAPP-A and free beta HCG and finite risk adding the ultrasound marker NT. The results show the effect of each marker in the formation of risk of Down’s syndrome, influence of biochemical markers on biochemical and finite risk and impact of NT marker on the final risk.

Table I

Influence of PAPP-A marker on biochemical risk.

	PAPP-A MOM normal value	PAPP-A MOM pathological value
Risk-free	98	86
Risk	68	88

Sensitivity 0.5563 (probability 55.63%)

Specificity of PAPP-A 0.4815 (probability 48.15%)

Positive expected value of PAPP-A 0.4615 (probability 46.15%)

Negative expected value of PAPP-A 0.5759 (probability 57.59%)

Table II

Influence of free beta HCG marker on biochemical risk.

	Normal value free beta HCG	Free beta HCG pathological value
Risk-free	109	77
Risk	63	91

Sensitivity of free beta HCG 0.5909 (probability 59.09%)

Specificity of free beta HCG 0.5860 (probability 58.60%)

Positive expected value of free beta HCG 0.5416 (probability 54.16%)

Negative expected value of free beta HCG 0.6337 (probability 63.37%)

Table III

Influence of PAPP-A marker on the final risk (biochemical + NT).

	PAPP-A MOM normal value	PAPP-A MOM pathological value
Risk-free	145	139
Risk	21	35

Sensitivity of PAPP-A 0.6250 (probability 62.50%)

Specificity of PAPP-A 0.5106 (probability 51.06%)

Positive expected value of PAPP-A 0.2011 (probability 20.11%)

Negative expected value of PAPP-A 0.8735 (probability 87.35%)

In the last two decades, there have been numerous reports about the detection rate for different methods of screening for trisomy 21. Detection rate of the risk of maternal age and fetal NT is 75–80%, while the risk for age and biochemical screening of PAPP-A and free beta HCG is 70%. The combination of age-related risk markers NT, PAPP-A and free beta HCG increases the detection of trisomy 21 to 85–95%

The ability to achieve a reliable measurement of NT is dependent on the appropriate training of sonographers

 

Biochemical analyzers provide auto mated, precise and reproducible measurements. Presenting selectively the biochemical and ultrasound screening in the first trimester and representing a separate risk of sonographic and biochemical markers give a much better insight than the pure presentation of their combination. This type of screening is achieved by a policy in which the first-stage screening is based on maternal age, fetal NT and either tricuspid or ductus venosus flow, and biochemical testing is then performed only in those with an intermediate risk. An alternative first trimester contingent screening policy consists of maternal serum biochemistry in all pregnancies followed by fetal NT only in those with an intermediate risk after biochemical testing

Double markers in fest trimester First trimester serum screening for abnormal proteins

  

 

Never thought in this way!!!!  Have you ever thought that serum amylates (Double markers-like serum free beta- hCG & PAPP-A) which represent foetal structural wellbeing to some extent comes from placenta and reflects placental wellbeing. I never thought in this way, Did you??

Maternal Blood Proteins in First Trimester Screening:-A number of proteins in the maternal circulation have been found during the time of pregnancy. Many of these proteins( Where from you are coming to maternal circulation ?? )  are made or modified by the placenta.

Differences in levels of some of the proteins have been observed in patients carrying a fetus with Down syndrome and certain other chromosome abnormalities. The discovery of these slight differences in protein levels forms the basis for using them in screening protocols.

These are referred to as biochemical markers. Certain patterns of biochemical markers have been associated with fetal Down syndrome as well as other conditions. The levels of these proteins change during pregnancy, so interpretation requires knowledge of the gestational age.

Also, the effectiveness of these proteins varies with gestational ages. For example, differences in protein levels may be observed during the second trimester but not the first, while other proteins show differences during the first trimester but not the second.

 

Q .1:  Should we routinely insist on “Prenatal screening for Down’s syndrome” in our country??

Ans:  Yes. As of now such tets i.e NT and Serum D markers are being almost routinely offered in the first trimester of pregnancy between 11 and 13.6 weeks. This includes A)  ultrasound measurement of nuchal translucency (NT) and  B) the determination of fetal maternal serum biomarkers like  pregnancy-associated plasma protein-A (PAPP-A) and free beta human chorionic gonadotropin (free beta-hCG).

Q.2: Question of feasibility in India?  One should remember that at India at a given point of time approximately 5% of population is pregnant. I intend to mean that pregnant women represent approximately 5% of the total population. Although one cannot expect that all women will seek antenatal care (100%), a realistic target should be set based on the current rate of coverage.

Q.3: Missing link!!!  Question of demand & supply.

An increase of 5 to 10% of antenatal care coverage per year could be contemplated if the current rate is 50% or less. What will happen to Sonologists working at India if there is 100% coverage for all preg women of India by proper standard ANC which includes NT scan .

I just can’t imagine the immense load of NT scan they have to carry out per day not to speak of supply of PPE for sonologist for each case of NT scan!!!