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The formation of catecholestrogens by 2- and 4-hydroxylation of monophenolic estrogens represents a major route of estrogen metabolism. In vitro and in vivo studies on catecholestrogens have shown that 2-hydroxylated catecholestrogens are primarily inactivated by O-methylation, while o-methylation of 4-hydroxylated estrogen is of minor importance. In the present study the in vivo production of isomeric 2- and 3-monomethyl ethers of 2-hydroxyestrogens was measured in 12 healthy omnivorous male volunteers aged 51 +/- 4 years. The sum of estrone and 17 beta-estradiol, 2-hydroxyestrogens (sum of 2-hydroxyestrone and 2-hydroxyestradiol), 4-hydroxyestrogens (sum of 4-hydroxyestrone and 4-hydroxyestradiol) and the sum of the isomeric monomethyl ethers of 2-hydroxyestrone and 2-hydroxyestradiol were measured in 24-h urinary samples. The determination included hydrolysis of steroid conjugates, separation by chromatographic steps and final quantification by radioimmunoassay. The specificity of the antibodies enabled differentiation between the isomeric monomethyl ethers. The mean urinary excretion rates were 8.8 +/- 2.9 micrograms/24 h for estrone plus estradiol, 5.2 +/- 2.4 micrograms/24 h for 2-hydroxyestrogens and 1.3 +/- 0.5 micrograms/24 h for the 4-hydroxyestrogens. The 2- and 3-monomethyl ethers of the 2-hydroxyestrogens were found in all individuals, with excretion rates of 5.8 +/- 2.6 micrograms/24 h for 2-methoxyestrogens and 3.6 +/- 1.1 micrograms/24 h for 2-hydroxyestrogen-3-methyl ethers. The findings indicated that 2-hydroxyestradiol is metabolized in vivo by 2-O-methylation and, to a lesser extent, by 3-O-methylation.

The long term ovariectomized adult rat was used to test the effects of exogenous estradiol, 4-hydroxyestradiol, and 2-hydroxyestradiol on LH secretion. To this end, different doses of estradiol 3-benzoate, 4-hydroxyestradiol 3,4-dibenzoate, and 2-hydroxyestradiol 2,3-dibenzoate were injected daily at 0800 h, and the LH serum levels were measured on 4 experimental days. At a dose of 1 micrograms/day, estradiol benzoate lowered LH secretion, beginning 48 h after the first injection (morning of day 2), and induced a characteristic LH surge 10 h later. 4-Hydroxyestradiol dibenzoate at the same dose produced less suppression of LH on the morning of day 3, but caused a comparable and highly significant surge on the same evening. Higher doses (3 and 10 micrograms/day) resulted in the same pattern seen with estradiol benzoate. 2-Hydroxyestradiol dibenzoate at comparable doses had no effect. Only extremely high doses (129 micrograms/day) caused slight suppression of tonic LH secretion 72 h after the first injection, and inconsistent LH elevations occurred on the same evening. It is concluded that in this model, catecholestrogens act as estrogens with respect to LH suppression and release, with 4-hydroxyestradiol being a potent estrogen and 2-hydroxyestradiol a weak estrogen.

A double isotope derivative determination technique was developed to investigate the excretion of 2-hydroxyestrone inhuman menstrual cycle. The method is highly specific, accurate and precise within the range of 0.2-20 nmol 2-hydroxyestrone/20ml of urine. The amounts of 2-hydroxyestrone excreted during menstrual cycle varied between 10 (proliferation phase) and 60 mug/24 h urine (ovulatory peak), which is comparable to that of estriol, supposed hitherto to be the main excretion product of estrogen metabolism.

4-Hydroxyestradiol-17 beta and 4-hydroxyestradiol-17 alpha (5 or 20 microgram/d) were continuously s.c. infused for 3 days into ovariectomized adult rats. The serum levels of either epimer were virtually identical when the same dose was administered. 4-Hydroxyestradiol-17 beta significantly altered body and uterus weight and LH serum levels (negative and positive effects) at both doses tested. 4-Hydroxyestradiol-17 alpha showed no effects even at the 20 microgram/d dose. As both epimers have similar affinities for catechol O-methyltransferase, but their potencies regarding effects on lH serum levels differ markedly, it is concluded that the interaction of catecholestrogens with this enzyme is not essential for their effects on LH release.

Urinary 2-hydroxyesterone was quantitatively determined in the course of several normal human pregnancies. The urine was subjected to hot acid hydrolysis and after chromatographic purification, 2-hydroxyesterone was converted into the corresponding phenazine derivative, which was submitted to a final column chromatogrphy and then quantitated by UV-spectrometry. For correction of procedural losses 2-hydroxyestrone-4-14C was used as internal standard. The urinary 2-hydroxyestrone of different subjects varied within a wide range especially at mid-pregnancy between 100 and 2500 mug/24 h. The day-to-day variation of the excretion of 2-hydroxyestrone was mostly less than 30% of the total value of that day, but sometimes could even reach 60%. The investigation of 2-hydroxyestrone and total estrogens at regular intervals throughout several pregnancies, showed that the excretion of 2-hydroxyesterone generally reached a maximum during the second trimester, while the excretion of the total estrogens steadily increased up to parturition. When analyzing the urines of different subjects during the last 4 months of pregnancy, no correlation appeared to exist for the excretions of the total estrogens and of 2-hydroxyestrone.

The effect of keoxifene (LY 156 758) on GnRH-stimulated LH release and its ability to antagonize estrogen actions were investigated in rat anterior pituitary cells. Estrogens exert either stimulatory or inhibitory effects on GnRH-induced LH secretion in rat pituitary cells depending on the incubation time with the steroid. When pituitary cells were treated for 24 h with 10(-9) M estradiol, the LH response to GnRH was clearly enhanced, and this effect was completely inhibited by 300 nM keoxifene. Short term treatment (4 h) of pituitary cells with 10(-9) M estradiol inhibits GnRH-stimulated LH release, and this effect was also blocked by keoxifene in a dose-dependent manner. In the absence of exogenous estrogen the treatment of pituitary cells for 4 h with increasing concentrations of keoxifene reduced the LH response to 10(-9) M GnRH only at very high concentrations (10(-5) M) of the antiestrogen. After treatment for 24 h, the inhibitory effect of keoxifene was evident at concentrations greater than or equal to 10(-8) M, with a reduction of GnRH-induced LH release by up to 60%. The effects of the antiestrogen were also analyzed in a dynamic culture system, in which pituitary cells grown on microcarrier beads were continuously perifused with medium and stimulated with GnRH in a pulsatile fashion. The LH response to a 2 min pulse of 10(-9) M GnRH was reduced in magnitude after 40 min of perifusion with 10(-9) M estradiol. When keoxifene (300 nM) was present at the same time, the LH response was identical to that observed in vehicle-treated cells. At the concentration of 300 nM, keoxifene per se did not change the responsiveness of the pituitary cells to the GnRH stimulus. These findings show that keoxifene is a potent antagonist of both positive and negative estrogen actions in the pituitary gonadotroph. In addition, after short term treatment with high concentrations or after long term treatment, keoxifene itself exerts an inhibitory effect on GnRH-induced LH secretion.

When [6,7-3H]estradiol was incubated with tissue homogenates of the brain, the pituitary, and the liver of two human female fetuses, a number of radioactive metabolites more "polar" than the incubated substrate were detected. Among these, the identification of two types of catecholestrogens, i.e. the 2- and 4-hydroxyestrogens, was of major interest. Compared on the basis of wet weight of tissues (250 mg), the conversion of estradiol to 2-hydroxyestrogens (2-hydroxyestradiol and 2-hydroxyestrone) was 0.8% in the frontal cortex, 1.0% in the hypothalamus, 2.1% in the pituitary, and 7.8% in the liver. For the first time, the formation of 4-hydroxyestrogens was demonstrated. The percentages of incubated estradiol hydroxylated at C-atom 4 (4-hydroxyestradiol and 4-hydroxyestrone) were 0.5 in the cortex, 0.4% in the hypothalamus, .1% in the pituitary, and 0.5% in the liver. The results show that fetal brain and pituitary tissue can hydroxylate estradiol in positions 2 and 4 to a similar extent, whereas in the liver, about 15 times more 2-hydroxy than 4-hydroxy compounds are formed. Moreover, the 2-hydroxylating capacity of the liver is definitely greater than that of the brain, whereas the 4-hydroxylating capacity is about the same as that of the brain.