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.     

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.     

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.     

Degradation of Estrogens by Rhodococcus zopfii and Rhodococcus equi Isolates from Activated Sludge in Wastewater Treatment Plants

We have isolated four strains of Rhodococcus which specifically degrade estrogens by using enrichment culture of activated sludge from wastewater treatment plants. Strain Y 50158, identified as Rhodococcus zopfii, completely and rapidly degraded 100 mg of 17β-estradiol, estrone, estriol, and ethinyl estradiol/liter, as demonstrated by thin-layer chromatography and gas chromatography-mass spectrometry analyses. Strains Y 50155, Y 50156, and Y 50157, identified as Rhodococcus equi, showed degradation activities comparable with that of Y 50158. Using the random amplified polymorphism DNA fingerprinting test, these three strains were confirmed to have been derived from different sources. R. zopfii Y 50158, which showed the highest activity among these four strains, revealed that the strain selectively degraded 17β-estradiol during jar fermentation, even when glucose was used as a readily utilizable carbon source in the culture medium. Measurement of estrogenic activities with human breast cancer-derived MVLN cells showed that these four strains each degraded 100 mg of 17β-estradiol/liter to 1/100 of the specific activity level after 24 h. It is thus suggested that these strains degrade 17β-estradiol into substances without estrogenic activity.     

The effect of oral contraceptive steroid therapy on the urinary metabolites of radioactive estrone and estradiol-17beta

Eight urinary metabolites of radioactive estrone and estradiol-17β (estrone, estradiol-17β, 2-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone 3-methyl ether, 16α-hydroxyestrone, 2-hydroxyestradiol and estriol) have been measured by reverse isotope dilution from young women on oral contraceptive therapy. There was a decrease in the sum of the 16α-hydroxy1ated metabolites in the Ketodase liberated fraction from the subjects taking ethynylestradiol containing preparations as compared to those taking preparations containing mestranol and those subjects who were taking no oral contraceptives. This report is also the first to document and measure 2-hydroxyestradiol as a urinary metabolite of radioactive estrone and estradiol.     

Biodegradation of pyrene by Mycobacterium frederiksbergense in a two-phase partitioning bioreactor system

Biodegradation of pyrene by Mycobacterium frederiksbergense was studied in a two-phase partitioning bioreactor (TPPB) using silicone oil as non-aqueous phase liquid (NAPL). The TPPB achieved complete biodegradation of pyrene; and during the active degradation phase, utilization rates of 270, 230, 139, 82 mg l(-1)d(-1) for initial pyrene loading concentrations (in NAPL) of 1000, 600, 400 and 200 mg l(-1), respectively, were obtained. The degradation rates achieved using M. frederiksbergense in TPPB were much higher than the literature reported values for an ex situ PAH biodegradation system operated using single and pure microbial species. The degradation data was fitted to simple Monod, logistic, logarithmic, three-half-order kinetic models. Among these models, only exponential growth form of the three-half-order kinetic model provided the best fit to the entire degradation profiles with coefficient of determination (R2) value >0.99. From the experimental findings, uptake of pyrene by the microorganism in TPPB was proposed to be a non-interfacial based mechanism.     

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。     

THE BIOGENESIS OF PRIMARY SEX HORMONES : I. THE FATE OF ESTRINS INJECTED INTO THE RABBIT

1. A method is given for the extraction and fractionation of rabbit urines which frees these urines of inactive chromogens but permits a quantitative recovery of estrone and estriol for the colorimetric determination of these compounds. 2. Estrone and estriol content of rabbit urine extracts can be determined by the concentration of the colored compound they form upon diazotization with sulfanilic acid and by the modified phenolsulfonic acid test of Cohen and Marrian. Estriol can be determined by the specific reaction first described by David. The technique for these tests is presented. 3. Estriol (300 micrograms) injected into rabbits (a) in heat, (b) pregnant, (c) pseudopregnant, (d) hysterectomized in heat, (e) hysterectomized pseudopregnant, (f) ovariectomized, is excreted in the urine as estriol. Rabbit does in the luteal phase (b, c, and e) excrete 3 to 4 times the amount of estriol excreted by females without corpora lutea (a, d, and f). 4. When estrone (300 micrograms) is injected into the same types of rabbit does types a, b, and c excrete both estrone and estriol, type f excretes both estrone and estriol shortly after ovariectomy, but only estrone at 2 months after castration. Hysterectomized animals (types d and e) never excrete estriol after estrone injection. The total urinary estrin (estrone plus estriol) in estrone-injected animals is increased 2 to 3 times in animals in the luteal phase (b, c, and e). 5. It is concluded that the uterus is the site of conversion of estrone to estriol, and that the conversion cannot take place in a uterus completely free of ovarian control (e.g., in long time ovariectomized animals). 6. In neither estrone-injected nor estriol-injected females is all the injected hormone recovered in the urine. The maximum recovery is 66 per cent. When estrone-benzoate (600 micrograms) is injected 94–98 per cent of the hormone is recovered from animals in the luteal phase (types c and e) and about 79 per cent in an ovariectomized female (type f). These data are taken to indicate that luteal secretions give partial protection against destruction to the hormones. 7. The observation that in certain of the urine extracts the hormone titer by bioassay is somewhat higher than the colorimetric titer may indicate that there is a slight conversion of estrone to estradiol, particularly since no equilenin was found in any of the extracts by colorimetric test. 8. The simultaneous injection of 300 micrograms of estrone and 500 micrograms of progesterone 4 days after an initial injection of 300 micrograms of estrone results in: (1) an increased estrin excretion in females in heat, hysterectomized unmated, and ovariectomized, and a slight decrease in the pseudopregnant female; (2) the appearance of estriol in the urine of the long time ovariectomized animal with no urinary estriol in a control ovariectomized animal receiving no progesterone. These findings are taken to prove that the conversion of estrone to estriol occurs in the uterus under the influence of progesterone. Since animals in heat produce small amounts of estriol after estrone injection it is inferred that the ovaries of estrus rabbits produce small amounts of corpus luteum hormone in the absence of formed corpora lutea.     

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.     

Degradation of estrogens by Rhodococcus zopfii and Rhodococcus equi isolates from activated sludge in wastewater treatment plants

We have isolated four strains of Rhodococcus which specifically degrade estrogens by using enrichment culture of activated sludge from wastewater treatment plants. Strain Y 50158, identified as Rhodococcus zopfii, completely and rapidly degraded 100 mg of 17beta-estradiol, estrone, estriol, and ethinyl estradiol/liter, as demonstrated by thin-layer chromatography and gas chromatography-mass spectrometry analyses. Strains Y 50155, Y 50156, and Y 50157, identified as Rhodococcus equi, showed degradation activities comparable with that of Y 50158. Using the random amplified polymorphism DNA fingerprinting test, these three strains were confirmed to have been derived from different sources. R. zopfii Y 50158, which showed the highest activity among these four strains, revealed that the strain selectively degraded 17beta-estradiol during jar fermentation, even when glucose was used as a readily utilizable carbon source in the culture medium. Measurement of estrogenic activities with human breast cancer-derived MVLN cells showed that these four strains each degraded 100 mg of 17beta-estradiol/liter to 1/100 of the specific activity level after 24 h. It is thus suggested that these strains degrade 17beta-estradiol into substances without estrogenic activity.     

UDPGA-dependent estrogen glucuronyltransferase of guinea-pig uterus: Assay,temporal relationships in pregnancy and some characteristics

The UDPGA-dependent estrogen glucuronyltransferase (GT) of guinea-pig uterus increases sharply at about 50 days of gestation to specific activity (SA) values which are, on average, 40-fold those during the first 7 weeks. The enzyme is endometrial and exhibits optimal activity at pH 8. Fetal, immature and adult non-pregnant animals possess low activity, or none at all. The uterine microsomal GT is activated by Mn²⁺, Ca²⁺ and Mg²⁺ and by low concentrations of the amphoteric detergent, Miranol H2M, but not by cholate, deoxycholate, Cutscum or Triton detergents. Miranol does not alter the k_M (estrone) of the enzyme. The SA of non-activated lever microsomal GT is 50-fold greater than that from uterus. The latter conjugates estrone and the isomeric estradiols but not estriol, testosterone, dehydroisoandrosterone, cortisol or p-nitrophenol. Activity toward estrone is inhibited by estradiol but not by estriol or p-nitrophenol. Liver GT conjugates estrone, estradiol, estriol, testosterone and p-nitrophenol. Uterine GT has apparent K_M values of 1.6 μM (estrone) and 0.2 mM (UDPGA). Liver microsomal activity shows a k_m of 2.4 μM for estrone. GT activity toward estrone is not present in guinea-pig placenta but is considerable in chorion and variable in amnion. It is speculated that the behaviour of the uterine enzyme is consistent with a control mechanism for uterine estrogen levels in late pregnancy.     

Biodegradation of a phenolic mixture in a solid–liquid two-phase partitioning bioreactor

A solid–liquid two-phase partitioning bioreactor (TPPB) in which the non-aqueous phase consisted of polymer (HYTREL) beads was used to degrade a model mixture of phenols [phenol, o-cresol, and 4-chlorophenol (4CP)] by a microbial consortium. In one set of experiments, high concentrations (850 mg l⁻¹ of each of the three substrates) were reduced to sub-inhibitory levels within 45 min by the addition of the polymer beads, followed by inoculation and rapid (8 h) consumption of the total phenolics loading. In a second set of experiments, the beneficial effect of using polymer beads to launch a fermentation inhibited by high substrate concentrations was demonstrated by adding 1,300 and 2,000 mg l⁻¹ total substrates (equal concentrations of each phenolic) to a pre-inoculated bioreactor. At these levels, no cell growth and no degradation were observed; however, after adding polymer beads to the systems, the ensuing reduced substrate concentrations permitted complete destruction of the target molecules, demonstrating the essential role played by the polymer sequestering phase when applied to systems facing inhibitory substrate concentrations. In addition to establishing alternative modes of TPPB operation, the present work has demonstrated the differential partitioning of phenols in a mixture between the aqueous and polymeric phases. The polymeric phase was also observed to absorb a degradation intermediate (arising from the incomplete biodegradation of 4CP), which opens the possibility of using solid–liquid TPPBs during biosynthetic transformation to sequester metabolic byproducts.     

Inhibitory effect of female hormones on lipid peroxidation

The female hormones estradiol, estrone, and estriol acted as antioxidants in the peroxidation of methyl linoleate by UV irradiation. All of them inhibited the peroxidation of microsomal lipids when they were added to the ADP-Fe3+ peroxidation system of rat liver microsomes. The efficiencies in the microsomal system were in the order of estradiol greater than estriol greater than estrone.     

Mandelic acid chiral separation utilizing a two-phase partitioning bioreactor built by polysulfone microspheres and immobilized enzymes

A novel two-phase partitioning bioreactor (TPPB) modified by polysulfone (PSF) microspheres and immobilized enzyme (novozym-435) was formed, and the resulting TPPB was applied into mandelic acid chiral separation. The PSF microspheres containing n-hexanol (named PSF/hexanol microspheres) was prepared by using the phase inversion method, which was used as the organic phase. Meanwhile, the immobilized enzyme novozym-435 was used as a biocatalyst. The water phase was composed of the phosphate buffer solution (PBS). (R, S)-Methyl mandelate was selected as the substrate to study enzymatic properties. Different reaction factors have been researched, such as pH, reaction time, temperature and the quantity of biocatalyst and PSF/hexanol microspheres added in. Finally, (S)-mandelic acid was obtained with an 80 % optical purity after 24 h in the two-phase partitioning bioreactor. The enantiomeric excess (ee_p) values were very low in the water phase, in which the highest ee_p value was only 46 %. The ee_p of the two-phase partitioning bioreactor had been enhanced more obviously than that catalyzed in the water phase.     

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.     

Biology and receptor interactions of estriol and estriol derivatives in vitro and in vivo

The biological effects of estriol (E3) have been studied in three estrogen targets, namely, the rat uterus in vivo and in vitro, in primary human endometrial cell cultures and in MCF-7 human breast cancer cells in culture. Studies on the temporal relationships between estrogen receptor binding and biological responses in the uterus using estriol and several more long-acting estriol derivatives, namely, 17α-ethynyl estriol, estriol-3-cyclopentyl ether, and 17α-ethynyl estriol-3-cyclopentyl ether, indicate that estriol is a short-acting compound with a brief duration of action. Estriol is a poor stimulator of uterine growth and plasminogen activator activity in vivo. Chemical modifications of the estriol molecule produce long-acting derivatives that result in a prolonged input of hormone receptor complexes into the nucleus and a prolonged and marked stimulation of uterine growth. In human endometrial cells in primary tissue culture, E₃ has 12% the affinity of estradiol (E₂) for cytosol estrogen receptor and it is quite effective yet slightly less potent than estradiol in stimulation of progesterone receptor synthesis. Low concentrations of E₃(10⁻¹⁰ M) stimulate growth of MCF-7 cells in vitro and dose-response curves show E₃ to be only slightly less effective than E₂. In these endometrial and breast cancer cell systems in vitro, there is no metabolism of E₃ while E₂ is metabolized to estrone.Hence, estriol is an effective estrogen in vitro. In vivo, it is short-acting, but it can be made a full estrogen agonist when given at a sufficiently high concentration or in a chemically modified form which prolongs its activity by enabling effective concentrations of the compound to be maintained in the blood and in target tissues.     

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.     

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.     

Estimating the cellular maintenance coefficient and its use in the design of two-phase partitioning bioscrubbers

One of the key roles of an organic solvent has emerged to be the enhancement of oxygen transfer in two-phase partitioning bioscrubbers (TPPBs). In order to determine an optimum organic fraction for a given VOCs loading, the oxygen demand of the total cell mass must be estimated, which depends upon the magnitude of the cellular maintenance coefficient. We have estimated the dynamics of the maintenance coefficient for benzene degradation by Achromobacter xylosoxidans Y234 in a TPPB and found that the maintenance coefficient generally decreased as cells accumulated in the TPPB but converged to a specific value of 1.750 × 10⁻² h⁻¹ at biological steady state. Due to its important influence on all of the essential design parameters of the TPPB system, including optimum organic fraction, aeration rate and agitation speed, the maintenance coefficient should be considered as a key biological determinant for microorganism selection, as well as in overall TPPB design.     

Biodegradation of estrogens by facultative anaerobic iron-reducing bacteria

Natural estrogens such as estrone, 17β-estradiol, estriol, and the synthetic component of contraceptive pills, 17α-ethinylestradiol, enter the municipal wastewater treatment plant via human excretions. A significant portion of these substances is found to remain in reject water produced after anaerobic digestion of activated sludge. In this study, the effect of the oxidant, Fe(III), and facultative anaerobic strain of iron-reducing bacteria on the anaerobic degradation of estrogens in reject water was investigated. Synthetic 17α-ethinylestradiol remained resistant to anaerobic biodegradation by iron-reducing bacteria, while natural estrogens such as 17β-estradiol, estriol, and estrone were removed by 92%, 60% and 27%, respectively, after 15 days of batch cultivation of iron-reducing bacteria in reject water with the addition of all estrogens to concentrations 100 μg l⁻¹ each. The ability of facultative anaerobic iron-reducing bacteria to degrade estrogens can be used for the anaerobic removal of trace organics from reject water in municipal wastewater treatment plant.