Estrogenic Mode Of Action Research Articles

Comparison of female rat reproductive effects of pubertal versus adult exposure to known endocrine disruptors.

A prevailing challenge when testing chemicals for their potential to cause female reproductive toxicity is the lack of appropriate toxicological test methods. We hypothesized that starting a 28-day in vivo toxicity study already at weaning, instead of in adulthood, would increase the sensitivity to detect endocrine disruptors due to the possibility of including assessment of pubertal onset. We compared the sensitivity of two rat studies using pubertal or adult exposure. We exposed the rats to two well-known human endocrine disruptors, the estrogen diethylstilbestrol (DES; 0.003, 0.012, 0.048 mg/kg bw/day) and the steroid synthesis inhibitor ketoconazole (KTZ; 3, 12, 48 mg/kg bw/day). Specifically, we addressed the impact on established endocrine-sensitive endpoints including day of vaginal opening (VO), estrous cyclicity, weights of reproductive organs and ovarian histology. After 28 days of exposure, starting either at weaning or at 9 weeks of age, DES exposure altered estrous cyclicity, reduced ovary weight as well as number of antral follicles and corpora lutea. By starting exposure at weaning, we could detect advanced day of VO in DES-exposed animals despite a lower body weight. Some endpoints were affected mainly with adult exposure, as DES increased liver weights in adulthood only. For KTZ, no effects were seen on time of VO, but adrenal and liver weights were increased in both exposure scenarios, and adult KTZ exposure also stimulated ovarian follicle growth. At first glance, this would indicate that a pubertal exposure scenario would be preferrable as timing of VO may serve as sensitive indicator of endocrine disruption by estrogenic mode of action. However, a higher sensitivity for other endocrine targets may be seen starting exposure in adulthood. Overall, starting a 28-day study at weaning with inclusion of VO assessment would mainly be recommended for substances showing estrogenic potential e.g., in vitro, whereas for other substances an adult exposure scenario may be recommended.

Chronic exposure of bisphenol S (BPS) affect hypothalamic-pituitary-testicular activities in adult male rats: possible in estrogenic mode of action

BackgroundThe industrial revolution has resulted in increased synthesis and the introduction of a variety of compounds into the environment and their potentially hazardous effects have been observed in the biota. The present study was aimed to evaluate the potential endocrine-disrupting effects of chronic exposure to the low concentrations of bisphenol S (BPS) in male rats.MethodsWeaning male Sprague-Dawley rats (22 days old) were either exposed to water containing 0.1% ethanol for control or different concentrations of BPS (0.5, 5, and 50 μg/L) in drinking water for 48 weeks in the chronic exposure study. After completion of the experimental period, animals were dissected and different parameters (hormone concentrations, histology of testis and epididymis, oxidative stress and level of antioxidant enzymes in the testis, daily sperm production (DSP), and sperm parameters) were determined.ResultsResults of the present study showed a significant alteration in the gonadosomatic index (GSI) and relative reproductive organ weights. Oxidative stress in the testis was significantly elevated while sperm motility, daily sperm production, and the number of sperm in epididymis were reduced. Plasma testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) concentrations were reduced and estradiol levels were high in the 50 μg/L-exposed group. Histological observations involved a significant reduction in the epithelial height of the testis along with disrupted spermatogenesis, an empty lumen of the seminiferous tubules, and the caput region of the epididymis.ConclusionThese results suggest that exposure to 5 and 50 μg/L of BPS for the chronic duration started from an early age can induce structural changes in testicular tissue architecture and endocrine alterations in the male reproductive system which may lead to infertility in males.

Determining ERβ Binding Affinity to Singly Mutant ERE Using Dual Polarization Interferometry

In a classic mode of estrogen action, estrogen receptors (ERs) bind to estrogen responsive element (ERE) to activate gene transcription. A perfect ERE contains a 13-base pair sequence of a palindromic repeat separated by a three-base spacer, 5′-GGTCAnnnTGACC-3′. In addition to the consensus or wild-type ERE (wtERE), naturally occurring EREs often have one or two base pairs’ alternation. Based on the newly constructed Thermodynamic Modeling of ChIP-seq (TherMos) model, binding energy between ER[Formula: see text] and a series of 34-bp mutant EREs (mutERE) was simulated to predict the binding affinity between ERs and EREs with single base pair deviation at different sites of the 13-bp inverted sequence. Experimentally, dual polarization interferometry (DPI) method was developed to measure ER[Formula: see text]–mutEREs binding affinity. On a biotin-NeutrAvidin (NA)-biotin treated DPI chip, wtERE is immobilized. In a direct binding assay, ER[Formula: see text]–wtERE binding affinity is determined. In a competition assay, ER[Formula: see text] was preincubated with mutant EREs before being added for competitive binding to the immobilized wtERE. This competition strategy provided a successful platform to evaluate the binding affinity variation among large number of ERE with different base mutations. The experimental result correlates well with the mathematically predicted binding energy with a Spearman correlation coefficient of 0.97.

The Effects Of Phthalate Exposure On Aromatase Expression In Adult Men

Introduction: The objectives of present study are to assess the associations among urinary phthalate metabolites levels, reproductive hormones and serum aromatase concentrations in adult men, and to explore the active mechanisms of phthalate exposure on aromatase expression in adult men. Methods: 298 subjects were recruited from the infertile clinic of National Cheng Kung University Hospital and AN-AN Women and Children Clinic, including 83 infertile men with abnormal semen quality (Group 1), 176 infertile men with normal semen quality (Group 2), and 39 fertile men (Group 3). After signed informed consent, the present study conducted through the questionnaire interview, determination of reproductive hormones, aromatase, and urinary phthalate metabolites. Urinary phthalate metabolites were analyzed with HPLC-MS/MS. Results: Urinary MMP, MEP, MiBP, MEHHP, MECPP, MEOHP, MINP and MIDP in Group 1 were higher than Group 2 and 3. Urinary MINP showed significant difference among three groups. Estradiol and serum aromatase concentrations in Group 1 were significant higher than Group 2 and 3 respectively. The significant increasing trends were found in estradiol levels and serum aromatase concentrations among the three groups respectively. After adjusting for age, BMI and season of blood drawing, urinary MMP, MEP and MEOHP were significantly positively associated with estradiol. Urinary MMP and MEP were significantly negatively associated with the TT:E2 ratio. Urinary MiBP, MnBP, MEHHP, MECPP, MEOHP were significantly positively associated with serum aromatase concentrations. The infertile men with abnormal semen quality had higher estrogen receptor binding effect and androgen receptor antagonistic effect than infertile men with normal semen quality and fertile men. Conclusion: phthalate exposure may increase serum aromatase concentrations, upregulation of CYP19a1 gene for the estrogen receptors, resulting in abnormally increase of estradiol levels by an estrogenic mode of action.

Immunohistochemical Localization of Estrogen Receptors in the Hippocampus of the Wistar Albino Rats.

Background: Estrogen plays an important role in changes taking place in the brain through the regulation of growth and differentiation of axons and dendrites, influence on plasticity, support of survival as well cognitive and behavioral functions. The classical mode of estrogen action is through the activation of its receptors, ER alpha and beta. ERα is more abundant in the arcuate nucleus, and ERβ is more prevalent in the hippocampus. Both receptors are located with the cell (genomic). Most of the literature are also supports the genomic ER importance and its role in cell signaling Purpose: To find out the genomic ER role and its cell signaling in hippocampus Methods and Materials: In this study extracellular neuronal ERs were identified in control as well as experimental group wistar albino rats by immunohistochemistry. Its importance in cell signaling was explained. In immunohistochemistry, indirect Avidin-Biotin Complex (ABC) formation method was used. Results: presence of genomic estrogen beta receptors in the hippocampus of wistar albino rats was measured. Presence of more number of in ER-IR pattern in (80.85±6.53) different subfields of CA as well as in DG by morphometric study. It suggest that presence of ERs through its estrogenic activity. These highly localized shifts in ER regulation and distribution may contribute to stress related decreases in synaptic plasticity While in this study confirms that more number of ER-IR was present within the presynaptic terminal and spines of CA1 pyramidal neurons of hippocampus. Conclusion: Genomic location of estrogen receptor in the hippocampus influence the hippocampal neuronal plasticity and cognition

Proliferation assays for estrogenicity testing with high predictive value for the in vivo uterotrophic effect

Proliferation assays based on human cell lines are the most used in vitro tests to determine estrogenic properties of compounds. Our objective was to characterise to what extent these in vitro tests provide alternatives for the in vivo Allen and Doisy test, a uterotrophic assay in immature or ovariectomised rodents with uterus weight as a crucial read-out parameter. In the present study four different human cell lines derived from three different female estrogen-sensitive tissues, i.e. breast (MCF-7/BOS and T47D), endometrial (ECC-1) and ovarian (BG-1) cells, were characterised by investigating their relative ERα and ERβ amounts, as the ERα/ERβ ratio is a dominant factor determining their estrogen-dependent proliferative responses. All four cell lines clearly expressed the ERα type and a very low but detectable amount of ERβ on both the mRNA and protein level, with the T47D cell line expressing the highest level of the ERβ type. Subsequently, a set of reference compounds representing different modes of estrogen action and estrogenic potency were used to investigate the proliferative response in the four cell lines, to determine which cell line most accurately predicts the effect observed in vivo. All four cell lines revealed a reasonable to good correlation with the in vivo uterotrophic effect, with the correlation being highest for the MCF-7/BOS cell line (R2=0.85). The main differences between the in vivo uterotrophic assay and the in vitro proliferation assays were observed for tamoxifen and testosterone. The proliferative response of the MCF-7/BOS cells to testosterone was partially caused by its conversion to estradiol by aromatase or via androstenedione to estrone. It is concluded that of the four cell lines tested, the best assay to include in an integrated testing strategy for replacement of the in vivo uterotrophic assay is the human MCF-7/BOS breast cancer cell line.

Predicting developmental toxicity through toxicogenomics

Global analysis of gene expression in target cells or tissues in response to a toxicant holds significant promise for predictive toxicology. Toxicants elicit a characteristic pattern of gene expression that is dependent on mechanism of action. These mechanism-specific transcript profiles can be used as the basis for predictive toxicology. Potential applications include prioritizing chemicals for testing and customizing testing approaches based on the chemical. Results that are useful in this predictive context can be obtained from animal or in vitro models. Gene expression analysis can also be used to elucidate the shape of the dose-response curve at exposure levels below the no observed adverse effect level, an important need in risk assessment. In this review, we will illustrate each of these points using our research on estrogen and an estrogenic mode of action as a model for how to use gene expression data in a predictive way. Although gene expression in response to estrogens is tissue, life stage, and sex specific, it is feasible to identify transcript profiles that are diagnostic of this mode of action.

Thresholds of toxicological concern for endocrine active substances in the aquatic environment

The threshold of toxicological concern (TTC) concept proposes that an exposure threshold value can be derived for chemicals, below which no significant risk to human health or the environment is expected. This concept goes further than setting acceptable exposure levels for individual chemicals, because it attempts to set a de minimis value for chemicals, including those of unknown toxicity, by taking the chemical's structure or mode of action (MOA) into consideration. This study examines the use of the TTC concern concept for endocrine active substances (EAS) with an estrogenic MOA. A case study formed the basis for a workshop of regulatory, industry and academic scientists held to discuss the use of the TTC in aquatic environmental risk assessment. The feasibility and acceptability, general advantages and disadvantages, and the specific issues that need to be considered when applying the TTC concept for EAS in risk assessment were addressed. Issues surrounding the statistical approaches used to derive TTCs were also discussed. This study presents discussion points and consensus findings of the workshop.

Estradiol, a key endocrine signal in the sexual differentiation and activation of reproductive behavior in quail

In Japanese quail, estrogen's effects on sexual behavior can be divided into three classes based on the underlying mechanisms and time-course of action and release. During embryonic life, the embryonic ovary secretes large amounts of estrogens. In contrast to what is observed in mammals where sexual differentiation essentially proceeds via masculinization of the males, in quail, females are demasculinized by their endogenous ovarian estrogens, an effect that can be blocked by injection of an aromatase inhibitor and mimicked in male embryos by an injection of estradiol. In adulthood, testosterone secreted by the testes is converted into estrogens by the preoptic aromatase. Locally produced estrogens activate male sexual behavior largely through the activation of estrogen receptors resulting in the transcription of a variety of genes, including brain aromatase (genomic effect). Both changes in estrogen production and action are observed within latencies ranging from a few hours to a few days, and are completely reversible. Additionally, brain aromatase activity can be modulated within minutes by calcium-dependent phosphorylations, triggered by variations in glutamatergic neurotransmission. These rapid changes in aromatase activity affect with relatively short latencies (10-15 min) the expression of male sexual behavior in quail and also in mice. Overall, the effects of estrogens on sexual behavior can thus be categorized into three classes: organizational (irreversible genomic action during ontogeny), activational (reversible genomic action during adulthood) and rapid nongenomic effects. Rapid and slower changes in brain aromatase activity match well with the two modes of estrogen action on behavior and provide temporal variations in the estrogens' bioavailability that should be able to support the entire range of established effects for this steroid.

Estrogen mobilizes a subset of estrogen receptor-alpha-immunoreactive vesicles in inhibitory presynaptic boutons in hippocampal CA1.

Although the classical mechanism of estrogen action involves activation of nuclear transcription factor receptors, estrogen also has acute effects on neuronal signaling that occur too rapidly to involve gene expression. These rapid effects are likely to be mediated by extranuclear estrogen receptors associated with the plasma membrane and/or cytoplasmic organelles. Here we used a combination of serial-section electron microscopic immunocytochemistry, immunofluorescence, and Western blotting to show that estrogen receptor-alpha is associated with clusters of vesicles in perisomatic inhibitory boutons in hippocampal CA1 and that estrogen treatment mobilizes these vesicle clusters toward synapses. Estrogen receptor-alpha is present in approximately one-third of perisomatic inhibitory boutons, and specifically in those that express cholecystokinin, not parvalbumin. We also found a high degree of extranuclear estrogen receptor-alpha colocalization with neuropeptide Y. Our results suggest a novel mode of estrogen action in which a subset of vesicles within a specific population of inhibitory boutons responds directly to estrogen by moving toward synapses. The mobilization of these vesicles may influence acute effects of estrogen mediated by estrogen receptor-alpha signaling at inhibitory synapses.

Histological and immunohistochemical evaluation of postmenopausal endometrium after 3 weeks of treatment with tibolone, estrogen only, or estrogen plus progestagen

To evaluate histological and immunohistochemical parameters of short-term (21 days) tibolone, estrogen-only, and estrogen+progestagen treatment in the human postmenopausal endometrium. An observational, open, nonrandomized, controlled study. Three collaborating centers: Amphia Hospital in Breda, Albert Schweitzer Hospital in Dordrecht, Erasmus Medical Center in Rotterdam, the Netherlands. Thirty healthy, postmenopausal women. Control group (n = 9), no hormonal treatment; tibolone group (n = 8), patients were treated with 2.5 mg of tibolone (administered orally) every day, starting 21 days before surgery; estrogen group (n = 7), patients were treated with 2 mg of E(2) (Zumenon, administered orally; Zambon, Amerfoort; The Netherlands) every day, starting 21 days before surgery; estrogen+progestagen group (n = 6), patients were treated with 2 mg of E(2) (Zumenon, administered orally) and 5 mg of medroxyprogesterone acetate (administered orally) every day, starting 21 days before surgery. Uterine tissues were collected, and two pathologists independently assessed histology. Immunohistochemical parameters measured were estrogen receptor alpha, progesterone receptor A/B, Hoxa10, Ki67, and Bcl-2. On the basis of a number of histological and immunohistochemical parameters measured after 21 days of treatment, it was observed that tibolone displays clearly less stimulation (proliferation) of the human postmenopausal endometrium than estrogen at the beginning of a treatment, but the stimulation is higher than with estrogen+progestagen. Short-term (21 days) tibolone treatment results in a small stimulation of proliferation of the endometrium, and because long-term treatment with tibolone has been demonstrated to lead to an atrophic endometrium, it may be concluded that the stimulatory effect, as observed in this study, is transient in nature. It is hypothesized that tibolone first displays a more estrogenic mode of action, which over time, is counterbalanced by the induction of its progestagenic properties.

Effects of chronic exposure to sodium arsenite on hypothalamo-pituitary-testicular activities in adult rats: possible an estrogenic mode of action.

BackgroundInorganic arsenic is a major water pollutant and a known human carcinogen that has a suppressive influence on spermatogenesis and androgenesis in male reproductive system. However, the actual molecular events resulting in male reproductive dysfunctions from exposure to arsenic remain unclear. In this context, we evaluated the mode of action of chronic oral exposure of sodium arsenite on hypothalamo-pituitary- testicular activities in mature male albino rats.MethodsThe effect of chronic oral exposure to sodium arsenite (5 mg/kg body weight/day) via drinking water without or with hCG (5 I.U./kg body weight/day) and oestradiol (25 micrograms oestradiol 3-benzoate suspended in 0.25 ml olive oil/rat/day) co-treatments for 6 days a week for 4 weeks (about the duration of two spermatogenic cycle) was evaluated in adult male rats. Changes in paired testicular weights, quantitative study of different varieties of germ cells at stage VII of spermatogenic cycle, epididymal sperm count, circulatory concentrations of hormones (LH, FSH, testosterone and corticosterone), testicular activities of delta 5, 3beta-hydroxysteroid dehydrogenase (delta 5, 3beta-HSD), 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), sorbitol dehydrogenase (SDH), acid phosphatase (ACP), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH), as well as the levels of biogenic amines (dopamine, noradrenaline and 5-hydroxytryptamine (5-HT)) in the hypothalamus and pituitary were monitored in this study. Hormones were assayed by radioimmuno- assay or enzyme- linked immunosorbent assay and the enzymes were estimated after spectrophotometry as well as the biogenic amines by HPLC electrochemistry.ResultsSodium arsenite treatment resulted in: decreased paired testicular weights; epididymal sperm count; plasma LH, FSH, testosterone and testicular testosterone concentrations; and increased plasma concentration of corticosterone. Testicular enzymes such as delta 5, 3 beta-HSD, 17 beta-HSD, and sorbitol dehydrogenase (SDH) were significantly decreased, but those of acid phosphatase (ACP), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) were significantly increased. A decrease in dopamine or an increase in noradrenaline and 5-HT in hypothalamus and pituitary were also noted after arsenic exposure. Histological evaluation revealed extensive degeneration of different varieties of germ cells at stage VII of spermatogenic cycle in arsenic exposed rats. Administration of human chorionic gonadotrophin (hCG) along with sodium arsenite partially prevented the degeneration of germ cells and enhanced paired testicular weights, epididymal sperm count, plasma and intratesticular testosterone concentrations, activities of delta 5, 3beta-HSD, 17 beta-HSD and sorbitol dehydrogenase along with diminution in the activities of ACP, ALP and LDH. Since many of the observed arsenic effects could be enhanced by oestradiol, it is suggested that arsenic might somehow acts through an estrogenic mode of action.ConclusionThe results indicate that arsenic causes testicular toxicity by germ cell degeneration and inhibits androgen production in adult male rats probably by affecting pituitary gonadotrophins. Estradiol treatment has been associated with similar effects on pituitary testicular axis supporting the hypothesis that arsenite might somehow act through an estrogenic mode of action.

Estrogen receptor-? is associated with the plasma membrane of astrocytes and coupled to the MAP/Src-kinase pathway

Estrogens influence CNS development and a broad spectrum of neural functions. Several lines of evidence also suggest a neuroprotective role for estrogen. Different modes of estrogen action have been described at the cellular level involving classical nuclear estrogen receptor (ER)-dependent and nonclassical membrane ER-mediated rapid signaling. We have previously shown that nonclassical estrogen signaling is implicated in the control of dopamine cell function and protection. Since nonclassical interactions between estrogens and glia may contribute to these effects, our aim was to demonstrate the presence of membrane-associated ERs and their putative coupling to intracellular signaling pathways in astrocytes. Confocal image analysis and fluorescence-activated cell sorting (FACS) studies indicated the attachment of ER-alpha but not ER-beta to the plasma membrane of astrocytes. ERs were located in the cell soma region and glial processes. FACS analysis revealed that only a subpopulation of midbrain astrocytes possesses membrane ER-alpha. In FACS studies on ER-alpha knockout astrocytes, only a few membrane ER-positive cells were detected. The activation of membrane ERs appears to be coupled to the MAP-kinase/Src signaling pathway as shown by Western blotting. In conclusion, our data provide good evidence that nonclassical estrogen action in astrocytes is mediated by membrane ER-alpha. The physiological consequence of this phenomenon is not yet understood, but it might have a pivotal role in estrogen-mediated protective effects on midbrain dopamine neurons.

Preoptic aromatase modulates male sexual behavior: slow and fast mechanisms of action

In many species, copulatory behavior and appetitive (anticipatory/motivational) aspects of male sexual behavior are activated by the action in the preoptic area of estrogens locally produced by testosterone aromatization. Estrogens bind to intracellular receptors, which then act as transcription factors to activate the behavior. Accordingly, changes in aromatase activity (AA) result from slow steroid-induced modifications of enzyme transcription. More recently, rapid nongenomic effects of estrogens have been described and evidence has accumulated indicating that AA can be modulated by rapid (minutes to hour) nongenomic mechanisms in addition to the slower transcriptional changes. Hypothalamic AA is rapidly down-regulated in conditions that enhance protein phosphorylation, in particular, increases in the intracellular calcium concentration, such as those triggered by neurotransmitter (e.g., glutamate) activity. Fast changes in brain estrogens can thus be caused by aromatase phosphorylation as a result of changes in neurotransmission. In parallel, recent studies demonstrate that the pharmacological blockade of AA by specific inhibitors rapidly (within 15–45 min) down-regulates motivational and consummatory aspects of male sexual behavior in quail while injections of estradiol can rapidly increase the expression of copulatory behavior. These data collectively support an emerging concept in neuroendocrinology, namely that estrogen, locally produced in the brain, regulates male sexual behavior via a combination of genomic and nongenomic mechanisms. Rapid and slower changes of brain AA match well with these two modes of estrogen action and provide temporal variations in the estrogen's bioavailability that can support the entire range of established effects for this steroid.

Induction of Proliferative Lesions of the Uterus, Testes, and Liver in Swiss Mice Given Repeated Injections of Sodium Arsenate: Possible Estrogenic Mode of Action

Inorganic arsenic (As) is a human carcinogen but has not been unequivocally proven carcinogenic in rodents. For instance, one older study indicates that repeated iv injections of sodium arsenate might induce lymphomas in Swiss mice (58% incidence) (Osswald and Goerttler, Verh. Dtsch. Ges. Pathol. 55, 289–293, 1971), but it was considered inadequate for critical evaluation of carcinogenic potential largely because of issues in experimental design. Therefore, we studied repeated iv sodium arsenate injection and neoplastic response in male and female Swiss mice. Groups (n = 25) of mice received sodium arsenate (0.5 mg/kg, iv) or saline (control) once/week for 20 weeks and were observed for a total of 96 weeks when the study ended. Differences in survival and body weights were unremarkable. In females, arsenate induced marked increases in the incidence and severity of cystic hyperplasia of the uterus compared against controls. Arsenate also was associated with a rare adenocarcinoma of the uterus. Hyperplastic uterine epithelium from arsenate-exposed animals showed strong positive immunostaining for the proliferating cell nuclear antigen (PCNA). There was also an upregulation of estrogen receptor (ER) immunoreactive protein in the early lesions of uterine luminal and glandular hyperplasia, although a progressive decrease in its expression was seen in the severe hyperplastic or neoplastic epithelium. In common with the preneoplastic and neoplastic gynecological lesions in humans, the levels of immunoreactive inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine-containing proteins were greater in the uterine hyperplastic epidermis and their intensity was positively correlated with the severity of the lesions. Arsenate-induced uterine hyperplastic lesions also showed a strong upregulation of cyclin D1, an estrogen-associated gene product essential for progression through the G1 phase of the cell cycle. In other tissues, arsenate increased testicular interstitial cell hyperplasia incidence and severity over control but without affecting the incidence of tubular degeneration. Arsenate also induced increases in hepatic proliferative lesions (HPL; foci of alteration + neoplasia), but only in females. Significant skin changes (incidence of hyperkeratotic lesions) and renal lesions (severity of nephropathy) also occurred in arsenate-treated females. Thus, repeated arsenate exposure, though not outright tumorigenic in the present study, was associated with proliferative, preneoplastic lesions of the uterus, testes, and liver. Estrogen treatment has been associated with proliferative lesions and tumors of the uterus, female liver, and testes in other studies, supporting a hypothesis that arsenate might somehow act through an estrogenic mode of action.

Mode of estrogen action on cell proliferative kinetics in CAMA-1 cells. I. Effect of serum and estrogen.

Both serum and estrogen affect cell proliferation in CAMA-1 cells. Their effects on cell cycle kinetics are being investigated with partially synchronized cells following 48 hours of serum deprivation. By comparing the growth kinetics of synchronized (serum-deprived) cells with asynchronized (normal-fed) cells, we observed that there was a delay of cell proliferation for approximately 25 hr for synchronized cells and that estrogen only induced cell proliferation in serum-supplemented culture. A serum protein (RPF), precipitated by ammonium sulfate, dialyzed in 3,500 daltons cut off membrane and reconstituted in culture medium, was shown to stimulate cell proliferation in a dose-related manner. In the absence of RPF, estrogen had no effect on cell growth and S-phase formation in serum-free medium, but significantly induced cell growth in the presence of RPF. Experiments conducted with a synchronized cell population showed that estrogen increased the proportion of G1 phase cells to enter S phase, shortened the G1 phase duration, and ultimately increased the proportion of mitotic cells per cycle. Collectively, these results show that (a) estrogen effects of cell proliferation in vitro requires a serum component, a "G1/S-promoting factor" and (b) estrogen-induced tumor growth is a result of an accelerated rate of G1/S transition and an increased number of dividing cells per cycle. The knowledge of the interaction of estrogen with the G1/S promoting factor on cell growth is of both fundamental and therapeutic importance.

Induction of lordosis in female rats: Two modes of estrogen action and the effect of adrenalectomy

In ovariectomized female rats, progesterone treatment alone does not induce lordosis, but following estrogen treatment by an appropriate interval it greatly enhances the performance of lordosis compared to that with estrogen alone. This “facilitating” effect of progesterone is thought to act synergistically with the initial “priming” effect of estrogen. In the present experiments a second estrogen treatment given to estrogen-primed ovariectomized rats in place of progesterone was found to facilitate lordosis. Latency of the facilitation of lordosis following this second estrogen treatment was similar to that of progesterone and was much shorter than that required for the usual “priming” effect, but higher doses were needed for the “facilitatory” effect. Experiments with adrenalectomized-ovariectomized female rats showed that this short latency effect of second estrogen treatment need not be mediated by the adrenals. These results raise the possibility that estrogen acts on the central nervous system in more than one way to induce lordosis.