The effects of estradiol and catecholestrogens on uterine glycogen metabolism in mink (Neovison vison)

Glycogen is a uterine histotroph nutrient synthesized by endometrial glands in response to estradiol. The effects of estradiol may be mediated, in part, through the catecholestrogens, 2-hydroxycatecholestradiol (2-OHE2) and 4-hydroxycatecholestradiol (4-OHE2), produced by hydroxylation of estradiol within the endometrium. Using ovariectomized mink, our objectives were to determine the effects of estradiol, 4-OHE2, and 2-OHE2 on uterine: 1) glycogen concentrations and tissue localization; 2) gene expression levels for glycogen synthase, glycogen phosphorylase, and glycogen synthase kinase-3B; and 3) protein expression levels for glycogen synthase kinase-3B (total) and phospho-glycogen synthase kinase-3B (inactive). Whole uterine glycogen concentrations (mean ± SEM, mg/g dry wt) were increased by estradiol (43.79 ± 5.35), 4-OHE2 (48.64 ± 4.02), and 2-OHE2 (41.36 ± 3.23) compared to controls (4.58 ± 1.16; P ≤ 0.05). Percent glycogen content of the glandular epithelia was three-fold greater than the luminal epithelia in response to estradiol and 4-OHE2 (P ≤ 0.05). Expression of glycogen synthase mRNA, the rate limiting enzyme in glycogen synthesis, was increased by 4-OHE2 and 2-OHE2 (P ≤ 0.05), but interestingly, was unaffected by estradiol. Expression of glycogen phosphorylase and glycogen synthase kinase-3B mRNAs were reduced by estradiol, 2-OHE2, and 4-OHE2 (P ≤ 0.05). Uterine phospho-glycogen synthase kinase-3B protein was barely detectable in control mink, whereas all three steroids increased phosphorylation and inactivation of the enzyme (P ≤ 0.05). We concluded that the effects of estradiol on uterine glycogen metabolism were mediated in part through catecholestrogens; perhaps the combined actions of these hormones are required for optimal uterine glycogen synthesis in mink.     

The sexually differentiated metabolism of [6,7-3H]17 beta-oestradiol in rats: male-specific 15 alpha- and male-selective 16 alpha-hydroxylation and female-selective catechol formation.

The oxygenated-metabolite profiles of exogenous 17 beta-oestradiol (E2) in adult male and female Wistar rats have been characterized and major sex-dependent biotransformations observed which correlate with the regioselectivities of known sexually differentiated hepatic P450. [6,7-3H]E2 (27 micrograms/kg) was given i.v. The metabolites of E2 were rapidly and extensively excreted in bile (46 and 78% of the dose over 1 and 6 h, respectively). Female rats metabolized E2 by one major pathway: oxidation to oestrone (E1) followed by C-2 hydroxylation and O-methylation; the principal aglycones (0-1 h bile collections) were E1 (14%), 2-hydroxyE1 (2-OHE1) (42%) and 2-methoxyE1 (24%). Male rats metabolized E2 principally by two parallel composite pathways of E1 hydroxylation which yielded a complex mixture of mono- and di-oxygenated compounds: 15 alpha-OHE1 (33%), 2,15 alpha-diOHE1 (7%), and 2-methoxy-15 alpha OHE1 (14%); 16 alpha-OHE1 (13%), 2,16 alpha-diOHE1 (4%) and 2-methoxy-16 alpha-OHE1 (2%). 15 alpha-Hydroxylation was unique to males. The balance of aromatic and alkyl hydroxylation in males was dose-dependent: at 3 mg/kg, 15 alpha-hydroxylation was decreased approx. 50% in favour of 2-hydroxylation whilst 16 alpha-hydroxylation was largely unaffected. The male-specific 15 alpha-hydroxylation and male-predominant 16 alpha-hydroxylation of E1 derived from E2 in vivo may be ascribable to the male-specific isoforms P450IIC13 and P450IIC11, respectively.     

The role of catecholestrogens in placental steroidogenesis

The effect of the catecholestrogen, 2-hydroxyestrone (2-OHE1), on placental steroidogenesis was studied by incubating 2-OHE1 with placental explants for 24 hours and measuring the output of estradiol (E2) and progesterone (P4). 2-OHE1 stimulated the accumulation of E2 and P4 in the media. This effect was inhibited by the alpha-adrenergic blocker, phenoxybenzamine, and the beta-adrenergic blocker, propranolol. We conclude that 2-OHE1 affects placental steroidogenesis and that this effect could possibly be mediated through adrenergic receptors.     

Cooxidation of steroidal and non-steroidal estrogens by purified prostaglandin synthase results in a stimulation of prostaglandin formation

Estrone (E1), estradiol (E2), the catechol estrogens 2-OHE1 and 2-OHE2, and diethylstilbestrol (DES) were incubated with purified prostaglandin synthase (PHS) in vitro in the presence of arachidonic acid and their PHS-catalyzed cooxidation was determined. 2-OHE1, 2-OHE2, and DES were extensively metabolized by PHS peroxidase activity, E1 and E2 to a lesser extent. The cooxidation of the estrogens is accompanied by an increased prostaglandin formation and an increase in cyclooxygenase activity in vitro; progesterone and nylestriol are without effect. Prostaglandins have been proposed to play a role in events related to early estrogen action in tissues such as the uterus. The cooxidation of estrogens and their metabolites by prostaglandin hydroperoxidase might represent one type of interaction between the hormones and the arachidonic acid cascade that could lead to changes in prostaglandins.     

17 beta-estradiol hydroxylation catalyzed by human cytochrome P450 1A1: a comparison of the activities induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells with those from heterologous expression of the cDNA.

Exposure of MCF-7 breast cancer cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes an elevated cytochrome P450 content and a marked increase in the microsomal hydroxylation of 17 beta-estradiol (E2) at the C-2, C-4, C-15 alpha, and C-6 alpha positions. In this study we investigated the involvement of cytochromes P450 of the 1A gene subfamily in this metabolism of E2. Hydroxylation at each of these four positions of E2 was inhibited by P450 1A-subfamily inhibitors, alpha-naphthoflavone, benzo[a]pyrene, and 7-ethoxyresorufin. Northern blots showed that treatment of MCF-7 cells with TCDD resulted in production of the 2.6-kb CYP1A1 mRNA, but not the 3.0-kb CYP1A2 mRNA. Immunoblot analyses with anti-P450 1A antibodies confirmed the production of P450 1A1 protein in TCDD-treated MCF-7 cells. Anti-rat P450 1A IgG inhibited the hydroxylation of E2 at C-2, C-15 alpha, and C-6 alpha, but not hydroxylation at C-4. E2 hydroxylation by human cytochromes P450 1A1 and P450 1A2 was assessed in experiments with microsomes from Saccharomyces cerevisiae after transformation with cDNAs encoding the two cytochromes. The major hydroxylase activities of expressed human P450 1A1 were at the C-2, C-15 alpha, and C-6 alpha positions of E2; expressed human P450 1A2 catalyzed hydroxylation predominately at C-2. While both expressed P450s 1A1 and 1A2 had minor hydroxylase activities at the C-4 position, neither catalyzed a low-Km hydroxylation at C-4 similar to that observed with microsomes from TCDD-treated MCF-7 cells. These results provide strong evidence that P450 1A1 catalyzes the hydroxylations of E2 at the C-2, C-15 alpha, and C-6 alpha in incubations with microsomes from TCDD-treated MCF-7 cells, but suggest TCDD may also induce a cytochrome P450 E2 4-hydroxylase that is distinct from P450 1A1 or P450 1A2.     

Is bisphenol A a weak carcinogen like the natural estrogens and diethylstilbestrol?

Bisphenol A (BPA) displays weak estrogenic properties and could be a weak carcinogen by a mechanism similar to that of estrone (E(1)), estradiol (E(2)) and the synthetic estrogen diethylstilbestrol, a human carcinogen. A wide variety of scientific evidence supports the hypothesis that certain estrogen metabolites, predominantly catechol estrogen-3,4-quinones, react with DNA to cause mutations that can lead to the initiation of cancer. One of the major pathways of estrogen metabolism leads to the 4-catechol estrogens, 4-OHE(1)(E(2)), which are oxidized to their quinones, E(1)(E(2))-3,4-Q. The quinones react with DNA to form predominantly the depurinating adducts 4-OHE(1)(E(2))-1-N3Ade and 4-OHE(1)(E(2))-1-N7Gua. This process constitutes the predominant pathway in the initiation of cancer by estrogens. One pathway of BPA metabolism is hydroxylation of one of its symmetric benzene rings to form its catechol, 3-OHBPA. Subsequent oxidation to BPA-3,4-quinone would lead to reaction with DNA to form predominantly the depurinating adducts 3-OHBPA-6-N3Ade and 3-OHBPA-6-N7Gua. The resulting apurinic sites in the DNA could generate mutations in critical genes that can initiate human cancers. The catechol of BPA may also alter expression of estrogen-activating and deactivating enzymes, and/or compete with methoxylation of 4-OHE(1)(E(2)) by catechol-O-methyltransferase, thereby unbalancing the metabolism of estrogens to increase formation of E(1)(E(2))-3,4-Q and the depurinating estrogen-DNA adducts leading to cancer initiation. Thus, exposure to BPA could increase the risk of developing cancer by direct and/or indirect mechanisms. Knowledge of these mechanisms would allow us to begin to understand how BPA may act as a weak carcinogen and would be useful for regulating its use.     

Monoclonal antibody-directed characterization of rat hepatic P450 catalyzing the omega-1 and omega-2 hydroxylation of prostaglandins

Previous studies have demonstrated that methylcholanthrene (MC) treatment of rats increases 10-fold the omega-2 hydroxylation of prostaglandin E2 (PGE2) by liver microsomes (K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477-489). The current study identifies the cytochrome P450 form, which catalyzes a major portion of the omega-2 hydroxylation of prostaglandins in liver microsomes of MC-treated rats (MC-microsomes) and examines whether the same enzyme catalyzes this reaction in microsomes from untreated rats (control microsomes). Three monoclonal antibodies (MAbs), MC 1-7-1, 1-31-2, and 1-36-1, raised against the major liver P450 from MC-treated rats were used. MAb 1-7-1 binds P450(57K) and P450(56K) (P450c and P450d, respectively); MAb 1-31-2 binds primarily P450(57K); and 1-36-1 binds solely P450(57k). MAb 1-7-1 inhibited omega-2 and omega-1 PGE2 hydroxylations in MC-microsomes by 70 and 45%, respectively. By contrast, MAb 1-31-2 and 1-36-1 were not inhibitory. MAb 1-7-1 did not inhibit PGE2 omega-2 hydroxylation in control or in microsomes from phenobarbital-treated rats (PB-microsomes). Since MAb 1-7-1 binds to both P450c and P450d, and 1-31-2 and 1-36-1 bind to P450c but are not inhibitory, these findings did not permit the determination of whether in MC microsomes a single isozyme (P450c or P450d) or both isozymes catalyze the omega-2 hydroxylation. This question was partially resolved by the observation that immunoaffinity-isolated P450c, supplemented with purified NADPH-P450 reductase, catalyzes effectively the omega-2 hydroxylation and to a lesser extent the omega-1 hydroxylation. There was no activity in the absence of reductase. The P450 antibody complex exhibits characteristics similar to those of the omega-2 hydroxylating activity in intact MC-microsomes supported by H2O2, by demonstrating a much higher activity when H2O2 is used instead of reductase and NADPH. Furthermore, a reconstituted monooxygenase composed of rat liver reductase and P450c, purified by conventional means, hydroxylated PGE2 at the omega-2 and omega-1 sites at a ratio of 2.8, similar to that obtained with the P450-antibody complex. These findings demonstrate that a major portion of the omega-2 hydroxylation of PGs in MC-microsomes is catalyzed by P450c; however, the possibility that some omega-2 hydroxylating activity is due to P450d was not ruled out.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytochrome P450 (CYP105F2) from Streptomyces peucetius and its activity with oleandomycin

The cytochrome P450 enzyme is one of the most versatile redox proteins and it is responsible for the oxidative metabolism of a wide variety of endogenous and exogenous compounds. The cytochrome P450 gene, CYP105F2, from Streptomyces peucetius was subcloned into the pET-32a(+) vector to overexpress the protein in E. coli BL21 (DE3) pLysS. The expressed enzyme was purified by fast protein liquid chromatography with a DEAE and UNO Q column. A 3D model was constructed based on the known crystallographic structures of cytochrome P450, and comparison with PikC and MoxA signified broad substrate specificity toward structurally diverse compounds. In addition, the in vitro hydroxylation of oleandomycin by purified CYP105F2 observed in liquid chromatography/mass spectrometry and mass/mass spectrometry indicated its flexibility towards alternative polyketides for the structural diversification of the macrolide by post-polyketide synthase hydroxylation.     

Cytochrome P-450C-M/F, a new constitutive form of microsomal cytochrome P-450 in male and female rat liver with estrogen 2- and 16 alpha-hydroxylase activity

A new cytochrome P-450 isozyme, P-450C-M/F, has been purified from untreated rat liver microsomes. The purified preparation was electrophoretically homogeneous and contained 12-15 nmol of P450/mg of protein and had a minimum molecular weight of 48,500. The NH2-terminal amino acid sequence of P-450C-M/F was different from that of other P-450's. Immunoblot analysis of microsomes demonstrated that P-450C-M/F was present in the liver of untreated male as well as female rats. Treatment of rats with phenobarbital, 3-methylcholanthrene, or beta-naphthoflavone did not induce P-450C-M/F. Cytochrome P-450C-M/F exhibited little activities of 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation or hydroxylation of arylhydrocarbon, testosterone, androstenedione, and progesterone. In contrast, it was highly active in N-demethylation of ethylmorphine and benzphetamine and in 2- and 16 alpha-hydroxylation of estrogens, particularly that of estradiol. These studies establish that cytochrome P-450C-M/F is constitutively present in both male and female rats and suggest that it may be involved in the oxidative metabolism of estradiol, particularly in the formation of estriol, the uterotropic metabolite of estradiol.     

Cytochrome P-450 isozymes from the marine teleost Stenotomus chrysops: their roles in steroid hydroxylation and the influence of cytochrome b5

Two new cytochrome P-450 forms were purified from liver microsomes of the marine fish Stenotomus chrysops (scup). Cytochrome P-450A (Mr = 52.5K) had a CO-ligated, reduced difference spectrum lambda max at 447.5 nm, and reconstituted modest benzo[a]pyrene hydroxylase activity (0.16 nmol/min/nmol P-450) and ethoxycoumarin O-deethylase activity (0.42 nmol/min/nmol P-450). Cytochrome P-450A reconstituted under optimal conditions catalyzed hydroxylation of testosterone almost exclusively at the 6 beta position (0.8 nmol/min/nmol P-450) and also catalyzed 2-hydroxylation of estradiol. Cytochrome P-450A is active toward steroid substrates and we propose that it is a major contributor to microsomal testosterone 6 beta-hydroxylase activity. Cytochrome P-450A had a requirement for conspecific (scup) NADPH-cytochrome P-450 reductase and all reconstituted activities examined were stimulated by the addition of purified scup cytochrome b5. Cytochrome P-450B (Mr = 45.9K) had a CO-ligated, reduced difference spectrum lambda max at 449.5 nm and displayed low rates of reconstituted catalytic activities. However, cytochrome P-450B oxidized testosterone at several different sites including the 15 alpha position (0.07 nmol/min/nmol P-450). Both cytochromes P-450A and P-450B were distinct from the major benzo[a]pyrene hydroxylating form, cytochrome P-450E, by the criteria of spectroscopic properties, substrate profiles, minimum molecular weights on NaDodSO4-polyacrylamide gels, peptide mapping and lack of cross-reaction with antibody raised against cytochrome P-450E. Cytochrome P-450E shares epitopes with rat cytochrome P-450c indicating it is the equivalent enzyme, but possible homology between scup cytochromes P-450A or P-450B and known P-450 isozymes in other vertebrate groups is uncertain, although functional analogs exist.     

Kinetics of catechol estrogen-estrogen receptor dissociation: a possible factor underlying differences in catechol estrogen biological activity

The mechanisms underlying the differences in uterotrophic potency between 2- and 4-hydroxyestrogens were explored. Doses of estradiol (E2)(10 micrograms/kg), 2-OHE2 (500 micrograms/kg) and 4-OHE2 (100 micrograms/kg) sufficient to induce near maximal cell nuclear estrogen receptor (ERn) binding were injected subcutaneously into 26 day old female rats. Uterine ERn concentrations declined more rapidly after 2-OHE2 than after E2 or 4-OHE2. E2 and 4-OHE2 both elicited a significant increase in uterine wet weight, measured at 24-36 hrs after injection. 2-OHE2 had no significant effect and neither synergized with nor antagonized the effects of simultaneously administered E2 or 4-OHE2. Under in vitro conditions at 25 degrees C, 2-hydroxyestrone (2-OHE1) and 2-OHE2 both dissociated from the receptors more rapidly than either their parent monophenolic estrogens or the corresponding 4-hydroxyestrogens. These results suggest that differences in estrogenic potency between 2- and 4-hydroxyestrogens may partly be a function of the dissociation kinetics of their estrogen receptor complexes.     

The first cytochrome P450 in ferns. Evidence for its involvement in phytoecdysteroid biosynthesis in Polypodium vulgare

The fern Polypodium vulgare is a phytoecdysteroid (PE)-producing plant. Cultures of P. vulgare prothalus produce PE, whereas prothalus-derived callus cultures do not. However, this callus line can transform topically applied ecdysone (E) to 20-hydroxyecdysone (20E), which is the last step in the biosynthetic pathway of the main plant PE. This hydroxylation is catalysed by a cytochrome P450 enzyme. E treatment of the callus line results in an increased amount of P450, showing a linear correspondence between the amount of P450 and in vivo E 20-hydroxylation activity, estimated by measuring the bioconversion of E to 20E. This activity can be inhibited by molecules that bind to the P450-heme group. E shows a P450-substrate-binding spectrum with microsomes that overexpress the P450 protein. Finally, a P450 protein was purified from E-treated calli, this being the first P450 to be described in the pterydophyte group.     

Steroidogenic response of rat and pig luteal cells to estradiol-17 beta and catecholestrogens in vitro.

The corpora lutea of several species contain estrogen receptors, but the role of estrogens in luteal function is unclear in most species. In this study, we investigated the direct effect of estradiol-17 beta (E2) and catecholestrogens (2-OHE2 or 4-OHE2) on rat and pig luteal steroidogenesis using in vitro cultures of small (SLC) and large (LLC) luteal cells prepared by elutriation. SLC and LLC were cultured at 37 degrees C for 36 h in serum-free media and treated with E2, 2-OHE2, or 4-OHE2; LH; forskolin (FORS); dibutyryl cAMP (dbcAMP); or combinations thereof. In the rat, E2 (2.5-10 micrograms/ml) inhibited progesterone (P4) production by both cell types dose-dependently. P4 production by rat SLC increased with increasing dose of 4-OHE2 up to the 2.5-microgram dose, then decreased to near control level at the 10-microgram dose. In LLC, P4 production in the presence of 4-OHE2 decreased initially (up to 2.5 micrograms/ml 4-OHE2), then increased at the 10-microgram dose. LH, FORS, and dbcAMP stimulated P4 production by SLC and LLC. For SLC, the stimulatory effects of LH and 4-OHE2 (2.5 micrograms) were comparable but lower than those of FORS and dbcAMP. For LLC, the effects of 4-OHE2 (10 micrograms), LH, and FORS were comparable but lower than those of dbcAMP. In time-course experiments, E2 inhibition of P4 production was observed at 36 and 72 h but not 6 h of culture for SLC and at all time points for LLC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preferential induction of cytochrome P450 1A1 over cytochrome P450 1B1 in human breast epithelial cells following exposure to quercetin

Estrogen metabolism is suggested to play an important role in estrogen-induced breast carcinogenesis. Epidemiologic studies suggest that diets rich in phytoestrogens are associated with a reduced risk of breast cancer. Phytoestrogens are biologically active plant compounds that structurally mimic 17beta-estradiol (E(2)). We hypothesize that phytoestrogens, may provide protection against breast carcinogenesis by altering the expression of estrogen-metabolizing enzymes cytochrome P450 1A1 (Cyp1A1) and 1B1 (Cyp1B1). Cyp1A1 and Cyp1B1 are responsible for the metabolism of E(2) to generate 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)), respectively. Studies suggest that 2-OHE(2) and 2-methoxyestradiol may protect against breast carcinogenesis, while 4-OHE(2) is carcinogenic in rodent models. Thus, agents that increase the metabolism of E(2) by Cyp1A1 to produce 2-OHE(2) may have chemoprotective properties. The human immortalized non-neoplastic breast cell line MCF10F was treated with quercetin at 10 and 50muM concentrations for time points ranging from 3 to 48h. Total RNA and protein were isolated. Real-time PCR was used to measure the expression of Cyp1A1 and Cyp1B1 mRNA. Quercetin treatment produced differential regulation of Cyp1A1 and Cyp1B1 mRNA expression in a time- and dose-dependent manner. Treatment with 10 and 50 microM doses of quercetin produced 6- and 11-times greater inductions of Cyp1A1 mRNA over Cyp1B1 mRNA, respectively. Furthermore, quercetin dramatically increased Cyp1A1 protein levels and only slightly increased Cyp1B1 protein levels in MCF10F cells. Thus, our data suggest that phytoestrogens may provide protection against breast cancer by modulating expression of estrogen-metabolizing genes such that production of the highly carcinogenic estrogen metabolite 4-OHE(2) by Cyp1B1 is reduced and the production of the less genotoxic 2-OHE(2) by Cyp1A1 is increased.     

Estradiol and catecholestradiols as possible genotoxic carcinogens

Estradiol and other estrogens are not classified as genotoxic carcinogens, but rather as tumor promoters in the multistage process of carcinogenesis. This study is a reexamination of the carcinogenic status of estradiol and the catecholestradiols (2-OHE2 and 4-OHE2) with the recently developed bacterial assays for genotoxic carcinogens, the Chromotest. The bacterial kits revealed estradiol and catecholestradiols as biphasic and potential genotoxic carcinogens with the following SOS inducing potency values: E2 43,265 (SD 8,300); 2-OHE2 30,153 (SD 2,500), and 4-OHE2 68,939 (SD 4,500). The differences between these values are statistically highly significant (p less than 0.0005). These results were confirmed by studies on Escherichia coli, which showed an increase in cell number and a stimulation of DNA content in the presence of the estrogens. It is therefore concluded that estradiol and the catecholestradiols are possible genotoxic carcinogens which probably act as tumor inducers rather than tumor promoters.     

Catecholestrogens excretion in smoking and non-smoking postmenopausal women receiving estrogen replacement therapy

Estrogens are involved in the etiology of breast cancer. Their blastomogenic influence may be partly realized through their conversion into catecholestrogens, rate of which may be modified by smoking. The risk of having breast cancer diagnosed can increase in women using estrogen replacement therapy (ERT). The principal aim of this investigation was to compare the excretion of classical estrogens and catecholestrogens in smoking and non-smoking postmenopausal women receiving Progynova (estradiol valerate, 2 mg/day, 1 month). Total 16 women were studied before and after treatment. Urinary estrogen profile method based on isotope dilution capillary gas chromatography-mass spectrometry was used. Before ERT, significantly lower excretion of 16-epiestriol and 4-hydroxyestrone (4-OHE1) and lower ratio of 4-OHE1/E1 were revealed in smokers. After ERT, much higher excretion of 2-OHE1, and 4-hydroxyestradiol (4-OHE2), higher ratios of 2-OHE1/E1 and 4-OHE1/E1 and lower ratio of 2-methoxyestrone/2-OHE1 were discovered in smokers as compared to non-smoking women. In conclusion only combination of ERT + smoking and not smoking itself leads to the specific prevalence of catecholestrogens (2-OH- and carcinogenic and DNA-damaging 4-OH-metabolites) that may increase risk of genotoxic variant of hormone-induced breast carcinogenesis without influence on the total morbidity.     

Effects of short-term exposure to catecholestrogens on serum concentrations of gonadotrophins and metabolism of catecholestrogens in ovariectomized rats

Equimolar amounts (36.8 nmol) of estradiol and the two catecholestrogens (CE's) 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol (2-OHE2) were injected subcutaneously to ovariectomized adult rats. Serum levels of both LH and FSH were determined at short-term intervals. Moreover, serum levels of the administered steroids and their main free and conjugated metabolites were monitored. Serum levels of the injected steroids reached peak values at different time points: estradiol between 60 and 240 min, CE's between 30 and 60 min. Peak height also differed significantly: with estradiol highest (1500 pg/ml), followed by 4-OHE2 (540 pg/ml) and then 2-OHE2 (135 pg/ml) (ratio 11:4:1). This mirrored the different MCR's of the steroids: CE's, especially 2-OHE2, were rapidly and extensively methylated and/or - to a lesser degree - conjugated. Estradiol remained mainly unchanged. LH-serum levels in steroid treated animals showed - irrespective of the steroid used - a uniform reaction pattern: they were significantly depressed 60 to 240 min after injection and - with the exception of estradiol treated rats - reached pretreatment levels again 480 min after injection. Ultra-short (15 min) effects were not observed. FSH serum levels in CE treated animals were not significantly altered, only E2 application led to a significant but small decrease in FSH levels 240 and 480 min after its injection. In conclusion, neither the effect of 4-OHE2 nor that of 2-OHE2 corresponded to the different MCR's or the MCR-corrected affinities for the classical estrogen receptor. A non-genomic mechanism may be responsible for this impaired effect of CE's.     

Heterologous expression, purification, and characterization of cytochrome P450sca-2 and mutants with improved solubility in Escherichia coli

Pravastatin, an important cholesterol lowering drug, is currently produced by hydroxylation of mevastatin (ML-236B) with Streptomyces carbophilus, in which the enzyme P450sca-2 plays a key role. Little information on the recombinant expression of this enzyme is available. As it is of industrial interest to develop an alternative simplified enzymatic process for pravastatin, as a first step, further study on the heterologous expression of this enzyme is warranted. We report here, for the first time, the purification, and characterization of P450sca-2 expressed in Escherichia coli. A synthetic gene encoding P450sca-2 was designed to suit the standard codon usage of E. coli. Expression of P450sca-2 in E. coli under optimized conditions yielded about 100 nmol purified active P450sca-2 per liter. Directed evolution was further carried out to improve the soluble expression level. In the absence of a facile and sensitive assay, green fluorescent protein (GFP) was used as a reporter to enable high-throughput screening. After three rounds of evolution by error-prone PCR and DNA shuffling, six almost totally soluble mutants were obtained, with the soluble expression levels dramatically improved by about 30-fold. For six most frequently occurring mutations, the corresponding single mutants were created to dissect the effects of these mutations. A single mutation, P159A, was found to be responsible for most of the enhanced solubility observed in the six mutants, and the corresponding single mutant also retained the hydroxylation activity. Our study provides a foundation for future work on improving functional expression of P450sca-2 in E. coli.     

Studies on the stoichiometry of estrogen oxidation catalyzed by purified prostaglandin-H-synthase holoenzyme

Prostaglandin-H-synthase (PHS) is a key enzyme in the biosynthesis of prostaglandins (PGs) from arachidonic acid and can oxidatively metabolize synthetic and steroidal estrogens. To investigate the relationship between estrogen cooxidation and PG synthesis, purified PHS-holoenzyme was incubated with radiolabeled arachidonic acid and various estrogens, namely diethylstilbestrol (DES), estradiol (E2), 2-hydroxyestradiol (2-OHE2), and 2-methoxyestradiol (2-MeOE2). The amount and pattern of PGs synthesized were analyzed by TLC and HPLC, estrogen metabolism was studied by HPLC. All tested compounds increased conversion of arachidonic acid to PG H2-derived prostanoids. A stoichiometric ratio between net estrogen oxidation and net PG H2 formation of approximately 2:1 for monophenolic compounds (2-MeOE2, E2) and of 1:1 for diphenolic estrogens (DES, 2-OHE2) was found, indicating that estrogens are apparently acting as electron donors for the PHS-peroxidase. In contrast, glutathione was not found to provide electrons for the reduction of PGG2 to PGH2, and rather decreased the conversion of arachidonic acid. The results of this in vitro study are discussed with respect to its implications for the in vivo situation.