Stimulation of estrogen receptor accumulation by estradiol in primary cultures of salmon hepatocytes

AbstractHepatocytes of Salmo salar in primary culture form confluent monolayers and can be maintained at 11 °C in serum-free medium for 8 days with minimal cell loss. Cultured hepatocytes from immature male salmon contain estrogen receptor both in nuclear and cytosol fractions (2000 and 2400 sites/cell, respectively). A single addition of estradiol results in an increase in the nuclear receptor to a level of 23 000 sites/cell after 24 h. This nuclear receptor concentration is similar to that in liver of estrogen-treated salmon in vivo, and is much higher than has been found for any other egg-laying vertebrate. The cultured salmon hepatocytes thus represent a highly sensitive system for the study of estrogen receptor dynamics and vitellogenesis in vitro

The oxidative metabolism of estrogens by mammalian liver

The main problem of estrogen metabolism studied has been to determine the nature of the water-soluble products formed from estrone-16-C¹⁴ by rat liver preparations. Comparative studies were carried out in the guinea pig. Three types of water-soluble metabolites were demonstrated, namely, protein-bound derivatives, glucosiduronate conjugates, and unidentified products which were not bound to protein and were not hydrolysed by 2N HC1. The water-soluble metabolites formed on incubating rat or guinea pig liver microsomes with estrone-16-C¹⁴ in the presence of NADPH and oxygen consisted of protein-bound material, some unknown derivatives, but virtually no simple conjugates. Incubation with the rat liver 8000 x g supernatant fraction resulted largely in conversion of the estrogen to the unknown water-soluble end-products, while in contrast, this liver fraction from guinea pig gave rise mainly to glucosiduronates. In the presence of UDPGA, both rat and guinea pig liver microsomes converted estrone-16-C¹⁴ to glucosiduronate conjugates, but this did not occur with the rat liver 8000 x g supernatant fraction. Estradiol-17β-16-C¹⁴and stilbestrol-C¹⁴ behaved similarly to estrone-16-C¹⁴. In the rat, in vivo, the bulk of the urinary water-soluble derivatives of estrone were of unknown nature, while in the guinea pig, glucosiduronate conjugation predominated. The problem was also studied by a different approach. Various compounds having structural features similar to estrone were tested for their ability to inhibit the formation of water-soluble metabolites from this estrogen by rat liver microsomes. It was found that 2-hydroxyestrone, 2-hydroxyestradiol-17β and equilenin were potent inhibitors, while those estrogens which had an oxygen function at C-6 or C-16, as well as the 17β-glucosiduronates and non-phenolic steroids tested were inactive. The synthetic estrogens, stilbestrol and hexestrol, both inhibited the reaction, but their non-estrogenic analogues had no effect. A group of benzoquinones, naphthoquinones and ortho- and para-hydroxylated phenols proved to be powerful inhibitors, whereas anthraquinones and meta-hydroxy-lated phenols showed no activity. In kinetic studies, 2-hydroxyestrone, equilenin, and stilbestrol appeared to act as competitive inhibitors, but menadione gave a mixed type of inhibition.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat