Biotransformation and Bioconcentration of Steroid Estrogens by Chlorella vulgaris

The biotransformation and bioconcentration of natural and synthetic steroid estrogens by Chlorella vulgaris were investigated by using batch-shaking experiments with incubation for 48 h in the light or dark. Estradiol and estrone were interconvertible in both light and dark conditions; however, this biotransformation showed a preference for estrone. In the light, 50% estradiol was further metabolized to an unknown product. Apart from biotransformation, estrone, as well as hydroxyestrone, estriol, and ethinylestradiol, was relatively stable in the algal culture, whereas estradiol valerate was hydrolyzed to estradiol and then to estrone within 3 h of incubation. All of the tested estrogens exhibited a degree of partitioning to C. vulgaris; however, the concentrations of estriol, hydroxyestrone, ethinylestradiol, and estradiol valerate were always below the quantification limits. For estradiol and estrone, the partitioning of these estrogens in the algal extracts to the filtrates was <6% of the total amount present. The average concentration factor for estrone was ca. 27; however, the concentration factor for estradiol was not reported since no equilibrium was reached between the aqueous solution and that within the cells due to continuing biotransformation.     

In vitro metabolism of (6,7-H3)estrone and of (6,7-H3) estradiol by the liver in pregnant guinea pigs]

Slices of pregnant guinea pig liver were incubated with (6,7-3H)estrone and with (6,7-3H)estradiol. Free, glucuro- and sulfo-conjugated fractions were isolated by specific extraction and hydrolysis. The radioactivity distribution in these 3 fractions demonstrated a predominance of conjugated compounds (95% of isolated estrogens) with slightly more glucuro-conjugated than sulfo-conjugated compounds. After isolating estrogens by TLC, we were able to determine estrone and estradiol in these 3 fractions from incubations with 3H-estrone or with 3H-estradiol by means of specific activity recrystallisation. Estriol was determined in glucuro-and sulfo-conjugated fractions after incubation with 3H-estrone as well as in sulfo-conjugated fraction after incubation with 3H-estradiol. Glucuro- or sulfo-conjugated estrone was the predominant estrogen after incubation with 3H-estrone just as after incubation with 3H-estradiol. This led us to conclude to an important 17beta-hydroxysteroid-dehydrogenase activity. The 16alpha-hydroxylastic-activity is weaker since estriol represented only 1,43 % of estrogens isolated after incubation with 3H-estrone and 0.82% after incubation with 3H-estradiol.     

Blood production rates of estrogens in women with differing ratios of urinary estrogen conjugates.

On the basis of the ratios of the estrogen conjugates in their urine (estriol/estrone + estradiol: E3/[E1+E2]), 19 women were divided into two groups: 9 women had ratios less than 0.6 and 10 women had ratios greater than 1.3. All women had measurements made of endogenous estrogens in their plasma by radioimmunoassay. They were then given constant infusions of 3H-estrone, 3H-estradiol and 14C-estriol during days 5-7 and days 20-22 of their cycles, and metabolic clearance rates (MCR) and blood production rates (PB) of estrone, estradiol and estriol were determined. Despite the wide disparity in their ratios of urinary estrogens, no differences could be found between the groups for the MCR's and PB's for all estrogens at either time of the cycle. The mean ratios of PB's (PB3/[PB2+PB1]) of estrone, estradiol and estriol ranged from 0.07 to 0.10 for each group during the cycle. The amounts of estriol entering the blood are small compared to the amounts of estrone and estradiol and do not correlate with the ratios of their urinary conjugates.     

Effects of estrone, estradiol and estriol combined with dihydrotestosterone on mounting and lordosis behavior in castrated male rats

Prepuberally castrated male rats were injected with estrone (1 or 5 μg), estradiol (1 or 5 μg) or estriol (1, 5, or 25 μg) either alone or in combination with dihydrotestosterone, (0.5 mg). Each of these steroids, when given alone, had no or only weak stimulatory effects on male sexual behavior. When combined with dihydrotestosterone all estrogens stimulated full copulatory behavior, the order of potency being estradiol, estrone, and estriol. Lordosis behavior in response to male mounting or manual stimulation was facilitated by all estrogens. All estrogens caused a slight weight increase of the seminal vesicles, ventral prostate and glans penis.     

Biodegradation of Endocrine Disruptors in Solid-Liquid Two-Phase Partitioning Systems by Enrichment Cultures

Naturally occurring and synthetic estrogens and other molecules from industrial sources strongly contribute to the endocrine disruption of urban wastewater. Because of the presence of these molecules in low but effective concentrations in wastewaters, these endocrine disruptors (EDs) are only partially removed after most wastewater treatments, reflecting the presence of these molecules in rivers in urban areas. The development of a two-phase partitioning bioreactor (TPPB) might be an effective strategy for the removal of EDs from wastewater plant effluents. Here, we describe the establishment of three ED-degrading microbial enrichment cultures adapted to a solid-liquid two-phase partitioning system using Hytrel as the immiscible water phase and loaded with estrone, estradiol, estriol, ethynylestradiol, nonylphenol, and bisphenol A. All molecules except ethynylestradiol were degraded in the enrichment cultures. The bacterial composition of the three enrichment cultures was determined using 16S rRNA gene sequencing and showed sequences affiliated with bacteria associated with the degradation of these compounds, such as Sphingomonadales. One Rhodococcus isolate capable of degrading estrone, estradiol, and estriol was isolated from one enrichment culture. These results highlight the great potential for the development of TPPB for the degradation of highly diluted EDs in water effluents.     

Estrogen determination using liquid chromatography with precolumn fluorescence labeling

A liquid chromatography procedure is described for the determination of some estrogens using fluorescence detection. The estrogens are labeled by precolumn derivatization with 5-dimethylaminonaphthalene-1-sulfonylchloride (dansyl chloride) and chromatographed on a reversed-phase, C-18 column with a mobile phase consisting of methanol, water, and acetic acid. The eluted analytes are measured with a fluorescence detector using excitation and emission wavelengths of 350 and 540 nm, respectively. The chief advantage of this new procedure is its sensitivity, requiring smaller amounts of sample to detect and quantitate estrogens in biological materials. We could detect less than 400 pg of estriol. With our procedure, this corresponded to about 25 ng in the final reaction mixture, before derivatization. The use of smaller sample volumes could improve this limit. Linearity for dansylated estriol, estrone, and estradiol was excellent over the estrogen range below 100 μg in the sample. This corresponds to approximately 1.7 μg on the column. Within-run precision was better than 5% for the full extraction and derivatization procedure for estriol from pregnancy urine samples. Chromatography is complete within 10 min for dansylated estriol, estrone, and estradiol.     

Quantitative evaluation of steroid extrogens by study of the vaginal epithelium thickness and mitotic index in mice

The potency of estrone, estradiol, estriol, and equilenin, administered to mice subcutaneously or intravaginally, was quantitated by vaginal mitotic index and by epithelial thickness; results were compared with those previously obtained in the classical tests of rat vaginal cornification and uterotrophic activity in mice. Ovariectomized mice received .002-.7 mcg estrogens in oil sc, or .16-6250 pg in alcohol solution intravaginally. 19 hours later .1 mg colchicine was given to arrest cells in metaphase. 24 hours after estrogen treatment, vaginas were a positive log-dose response in both tests and by both routes. Estradiol by both routes increased vaginal thickness but without linear dose-response, increased vaginal mitosis with a less definite dose response, but generated a negative dose response in mitotic index. Equilenin had a positive but nonlinear effect by both routes in both tests. Comparing the activities of these estrogens by routes, estradiol was more active by subcutaneous than by intravaginal routes; estriol and equilenin were more active vaginally than subcutaneously. Estradiol was 3-7 times more active than estrone intravaginally and 25-45 times more active subcutaneously. Estriol was less active than estrone; equilenin was as active as estrone intravaginally, but less active subcutaneously. In comparison with the rat vaginal cornification or mouse uterotrophy tests, estradiol sc, estriol and equilenin sc and especially vaginally, are much more active.     

A sensitive determination of estrogens with a Pt nano-clusters/multi-walled carbon nanotubes modified glassy carbon electrode

On the top of a multi-walled carbon nanotubes (MWNTs) modified glassy carbon electrode (MWNTs/GCE), Pt nanoclusters were electrochemically deposited, fabricating a Pt/MWNTs composite modified electrode, Pt/MWNTs/GCE. X-ray photoelectron spectroscopy, powder X-ray diffraction and field emission scanning electron microscope were used for the surface characterization of the electrode, and demonstrated the formation and distribution of Pt clusters of Pt nanoparticles of 8.4 nm in averaged size in the MWNTs matrix. The preliminary study found that this composite modified electrode has strong electrocatalytic activity toward the oxidation of estrogens involving estradiol, estrone and estriol. The voltammetric behavior of estrogens on this electrode was investigated by cyclic voltammetry, linear sweep voltammetry and square-wave voltammetry. In comparison with the MWNTs/GCE or a Pt nanoparticles modified GCE prepared in the similar way, this composite modified electrode exhibited much higher current sensitivity and catalytic activity. This electrode is also stable. The linear range of square-wave voltammetric determination was 5.0 x 10(-7)-1.5 x 10(-5)mol/L for estradiol, 2.0 x 10(-6)-5.0 x 10(-5)mol/L for estrone, and 1.0 x 10(-6)-7.5 x 10(-5)mol/L for estriol. Under an assumption that the concentration ratio of estradiol:estrone:estriol is 2:2:1, the real sample of blood serums was tested for the determination using this electrode. Satisfactory result was obtained with averaged recovery of 105%.     

Substrate dependency of specific and non-specific estrogen-2/4-hydroxylase activities measured by the radio-enzymatic method in rat brain microsomes

Putative specific and non-specific estrogen-2/4-hydroxylase activities which might affect the radio-enzymatic assay were characterized in terms of their requirements for NADPH and their substrate dependency. Using rat brain microsomes and partially purified rat liver COMT three main sources of estrogen-2/4-hydroxylase activity could be distinguished; a COMT-related component and NADPH-dependent and NADPH-independent microsomal components. The COMT-related activity required NADPH and showed about equal preferences for estrone and estradiol. The NADPH-dependent component was highly specific for estradiol, the relative activities observed with estrone and estriol being 7 and 1% of that observed with estradiol. The NADPH-independent component exhibited substrate saturation, was heat-labile and could not be inhibited by alpha-naphthoflavone or metyrapone. It showed a preference for estrone over estradiol, with estriol being a very poor substrate. These findings indicate that non-enzymatic factors contribute very little to product formation in the radio-enzymatic assay. The specificity of the major NADPH-dependent microsomal component towards estradiol suggests a stereo-specific requirement for the D-ring configuration of this estrogen. The use of no-cofactor blanks in the radio-enzymatic assay may be very important when different estrogens are compared as substrates for estrogen-2/4-hydroxylases.     

Estrogen metabolism in nonhuman primates I. In vitro biosynthesis of estrogen glucosiduronates in rhesus monkey liver

Estrone glucosiduronate, 17β-estradiol-3-glucoslduronate, 17β-estradiol-17-glucosiduronate and estriol-16α-glucoslduronate have been biosynthesized in substantial yield by incubation of radioactive estrone, 17β-estradiol, estriol and uridlne diphosphoglucosiduronic acid with rhesus monkey liver homogenates. The metabolites were characterized by chromatography on Celite and DEAE-Sephadex, enzyme hydrolysis, derivative formation and crystallization to constant specific activity. The percent conversion to 17β-estradiol-17-glucosiduronate from 17β-estradlol ranged between 56–71%; from estrone, the conversion was 49–54%. Other metabolites formed from estradiol were estrone glucosiduronate(12–21%) and 17β-estradiol-3-glucosiduronate(5–12%). The same metabolites derived from estrone accounted for 18–28% and 10–14% respectively. After estriol incubation, more than 90% of the steroid was converted to estriol-16α-glucosiduronate with no detectable estriol-3-glucosiduronate. This report represents the first time that 17β-estradiol-17-glucosiduronate has been reported as a metabolite in the rhesus monkey and this is the only known species which forms 17β-estradiol-17-glucosiduronate as the predominant metabolite of either estrone or estradiol $\text{in vitro}$.Rhesus monkey liver is similar to the human and baboon in that it metabolizes estriol exclusively to estriol-16-glucosiduronate.     

Estrogen biosynthesis in human liver--a comparison of aromatase activity for C-19 steroids in fetal liver, adult liver and hepatoma tissues of human subjects

After incubation of various tritiated C-19 steroids (androstenedione, testosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate) with human fetal liver, adult liver and hepatoma tissue homogenates, estrone, estradiol and estriol were analysed after a series of purification steps involving column chromatography, thin layer chromatography and co-crystallization. The findings indicated that the human fetal liver extensively aromatized various C-19 steroids to estrogens, whereas human adult liver and hepatoma tissues exhibited little or no aromatase activities. The formation of estradiol from androstenedione in human fetal liver indicated the presence of 17 beta-hydroxysteroid dehydrogenase in this tissue. It was therefore concluded that although the liver participated in the aromatization process during the fetal stage, extensive aromatization did not take place in the adult liver.     

Estrone and estradiol metabolism in vivo in human breast cysts

Fibrocystic disease of the breast manifesting palpable cysts express breast cyst fluids frequently containing estrogen sulfates at concentrations far exceeding those found in sera of the patient. The study explored the potential of the breast cyst to synthesize some of these estrogen sulfates. Deuterated estrone and estradiol were synthesized and either (estradiol, 4 cases or estrone, 2 cases) was injected into a cyst. The cyst was aspirated at approximately 0, 4 and 8 h, the target being 1 ml, 50% and complete aspiration respectively. Metabolites were purified sequentially by ether extraction, enzymatic hydrolysis of estrogen conjugates, chromatography on Sephadex LH 20 and identified by gas chromatography linked to mass spectrometry. The unconjugated fraction isolated from the ether extract was subjected to the same purification and detection scheme. Among the conjugates, deuterated estrone sulfate was the major metabolite of either precursor in all studies, while estradiol sulfate was not detected in any of the 6 experiments. The sulfate fractions also yielded traces of 16alpha-hydroxyestrone (2 studies), 4-hydroxyestrone (4 studies) and 2-hydroxyestrone (1 study). In the unconjugated fraction, one study with deuterated estradiol, 4- hydroxyestrone was obtained. In one study with deuterated estrone, traces of 2-hydroxyestrone and 16alpha- hydroxyestrone were obtained. These novel data are significant because patients with fibrocystic disease are at slightly elevated risk for developing breast cancer and 16alpha-hydroxyestrone and 4- hydroxyestrone are reported carcinogens.     

Metabolism of E1 and E2 in Ishikawa endometrium carcinoma cells: influence of TNF alpha

The metabolism of estrone (E1) and estradiol (E2) by Ishikawa endometrial carcinoma cells and its alteration by TNF alpha treatment was studied. Whereas this cell line practically does not respond to estrogens, it is very sensitive to TNF with respect to growth inhibition and other parameters. E2 and E1 were found to be metabolized by this cell line whereby the main metabolite for both estrogens was estriol. TNF significantly increased the rate of E1 and E2 conversion.     

Novel and potent biological antioxidants on membrane phospholipid peroxidation: 2-hydroxy estrone and 2-hydroxy estradiol

Catechol estrogens, 2-hydroxy estrone, 2-hydroxy estradiol and 2-hydroxy estriol, were tested as possible antioxidants of phospholipid peroxidation induced by Fe3+-ADP-adriamycin, using phospholipid liposomes as lipid source and alpha-tocopherol or other steroids as reference compounds. The parameters of antioxidant activities were: elongation of induction period, inhibition of O2 consumption required for lipid peroxidation and inhibition of peroxidative cleavage of unsaturated phospholipid. Of the tested compounds, 2-hydroxy estradiol or 2-hydroxy estrone had more potent activity than that of tocopherol.     

A kinetic study on the lactoperoxidase catalyzed oxidation of estrogens

Lactoperoxidase, which is produced in mammary glands, is proposed to be involved in carcinogenesis, because of its ability to react with estrogenic molecules, oxidizing them to free radicals. In the present study the reactivity towards six species (estradiol, ethynylestradiol, estriol, estrone, pregnenolone and mestranol) was investigated by means of a NADH-coupled system. The enzyme activity towards estradiol, ethynylestradiol, estriol and estrone did not vary much, suggesting that the different substituents in the D-ring of the steroid had little effect on the reaction. A somewhat higher K _m-value was obtained with estriol; possibly because of a more effective splitting of the enzyme–substrate complex into products. Pregnenolone, without resonance in the A-ring, and a methyl group in 19-position, did not react with the enzyme, in spite of having the proposed essential hydroxyl group in 3-position. Mestranol, with a methoxy group in 3-position, did not react with the enzyme either, supporting the suggestion that lactoperoxidase reacts with the 3-hydroxyl group of the estrogens.     

Luteinizing hormone concentrations in anestrous ewes administered various estrogens

Twenty four anestrous ewes were evenly assigned to one of six groups and administered either sesame oil, estradiol-17β, estradiol-17α, estrone, estradiol benzoate or estradiol valerate. All estrogen treated ewes received 50 μg of the respective estrogen. Blood plasma was collected for 28 hours post-treatment and quantified for luteinizing hormone (LH) by radioimmunoassay. An estrogen induced LH surge was detected in at least three of the four ewes administered either estradiol-17β, estrone, estradiol benzoate or estradiol valerate whereas only one of the four estradiol-17α treated ewes and none of the ewes administered sesame oil had an LH surge. The interval from treatment to peak LH was similar for estradiol-17β (17.3±2.7 hours), estrone (18.5±1.0 hours) and estradiol benzoate (19.0±0.6 hours) treated ewes but delayed 7 to 9 hours for ewes administered estradiol valerate (26.0±1.2 hours).     

畜禽养殖过程中雌激素的排放及其环境行为

由于存在广泛和较强的内分泌干扰性,环境雌激素越来越受到关注,其中人与动物排放的天然类固醇雌激素(雌酮、雌二醇和雌三醇)具有最强的干扰性。综述了畜禽养殖过程中天然雌激素的排放、危害以及其物化性质,并结合国内外近期研究阐明了天然雌激素的吸附、降解和迁移转化等环境行为。在目前雌激素研究现状的基础上,对未来的研究方向及目标提出了建议。     

Influence of different estrogens on neuroplasticity and cognition in the hippocampus

Background

Estrogens modulate the morphology and function of the hippocampus. Recent studies have focused on the effects of different types of estrogens on neuroplasticity in the hippocampus and cognition. There are three main forms of estrogens found in mammals: estradiol, estrone, and estriol. The vast majority of studies have used estradiol to investigate the effects of estrogens on the brain.

Scope of review

This review focuses on the effects of different estrogens on adult hippocampal neurogenesis, synaptic plasticity in the hippocampus, and cognition in female rats.

Major conclusions

Different forms of estrogens modulate neuroplasticity and cognition in complex and intriguing ways. Specifically, estrogens upregulate adult hippocampal neurogenesis (via cell proliferation) and synaptic protein levels in the hippocampus in a time- and dose-dependent manner. Low levels of estradiol facilitate spatial working memory and contextual fear conditioning while high levels of estradiol impair spatial working, spatial reference memory and contextual fear conditioning. In addition, estrone impairs contextual fear conditioning.

General significance

Advances in our knowledge of how estrogens exert their effects on the brain may ultimately lead to refinements in targeted therapies for cognitive impairments at all stages of life. However caution should be taken in interpreting current research and in conducting future studies as estrogens likely work differently in males than in females.     

Monitoring ovarian function in marmosets and tamarins by the measurement of urinary estrogen metabolites

Practical aspects of urinary estrogen analysis were considered with regard to establishing simple and reliable methods for monitoring ovarian function in marmosets and tamarins. Changes in the hormone:creatinine ratio in small volumes of urine from the common marmoset were significantly correlated with changes in 24-h excretion. Comparison of the metabolism and excretion of estrogens during the ovarian cycle in the common marmoset and cottontop tamarin revealed interesting species differences. High concentrations of conjugated estrone were measured in marmoset plasma, but estradiol 17β was the predominant estrogen in urine. In contrast, estrone was the most abundant estrogen measured in tamarin urine. Both species excreted very little estriol. Sulfates and glucuronides were present in urine in similar proportions before ovulation in the marmoset, although after ovulation sulfates were the more abundant. Conversely, most of the estrogens in tamarin urine appeared to be conjugated as glucuronides. Direct assay for estrone sulfate was applied to the measurement of urinary estrogen excretion during the ovarian cycle in a marmoset. The results compared well with those for total estradiol 17β after hydrolysis and ether extraction. The use of direct assays for conjugated estrogens in small volumes of urine is suggested as a practical method for monitoring ovarian function in marmosets and tamarins.     

The comparative potency of various steroids to complete the priming process for lordosis in guinea pigs

Ovariectomized adult guinea pigs were treated with a regimen of estradiol benzoate (0.2 μg/animal estradiol benzoate at hr 0 and 19) that was shown to be minimally effective for the induction of lordosis. They were then treated with 10, 20, or 80 mg of enclomiphene, 5, 20, 40, or 100 μg of estradiol, or testosterone, cortisol, estrone, estriol, diethylstilbestrol, catechol estradiol, or catechol estrone (all at a dose equivalent to 5 μg of estradiol) at hr 28. At hr 39 all females were given 0.5 mg progesterone, and subsequently tested for lordosis behavior. Of the various agents injected at hr 28 only estradiol (at all doses given), estrone, estriol, and diethylstilbestrol were effective in supporting display of lordosis behavior. The results indicate that the antiestrogen enclomiphene, the catechol estrogens, and at least some C₁₉ and C₂₁ steroids are weaker than E₂ or ineffective in facilitating lordosis behavior when given late in the priming period. Because previous work had shown that enclomiphene has partial estrogenic effects on lordosis behavior when administered early in the priming period (i.e., at hr 0, 19), it is suggested the early and late phases of the priming process induced by E₂ entail qualitatively different neural processes.