MECHANISM OF INACTIVATION OFα-ESTRADIOL BY RAT LIVER “IN VITRO”¹

Abstract

IT IS WELL known that the liver is the most important site of estrogen inactivation in different species. The early work of has shown that 95 per cent or more of the biological activity disappears when estrone is administered to man or rats or is incubated with rat liver "brei." Heller (1940) studied the inactivation of estrone, estradiol and estriol by slices of various tissues and the influence of several inhibitors upon this phenomenon. Engel and Rosenberg (1945) have observed that aqueous liver extracts, obtained at different pH, inactivate estrone in prolonged incubation. More recently Levy (1947), working with a liver extract prepared by a modification of technique, has been able to study several properties of the inactivating system.The present study was undertaken for the purpose of securing additional information regarding the nature of the estrogen-inactivating mechanism. METHODS AND TECHNIQUESMale Wistar descendants, weighing from 150 to 300 gm. were used throughout. Different types of liver preparations were employed. Rats were killed by a blow on the head and section of the neck with scissors. The liver was immediately removed and was either sliced according to the well known Warburg technique or subjected to the following procedures:Three other preparations were used, i.e., frozen powder, homogenate and acetone powder.(1) The powder was prepared by freezing liver with a mixture of dry ice and ethyl alcohol and pulverizing the hardened tissue in a mortar. A 10 per cent suspension of the powder in Krebs (1933) solution was used for the experiments.(2) A 10 per cent homogenate was prepared. The required amount of liver was dropped in an ice-cold suspending medium, previously placed in the glass container of a Waring Blendor. The glass container was kept cold by packing with crushed ice and the tissue was homogenized for 3 minutes in a cold room at approximately 5° C.

The Chemistry and Metabolism of the Steroid Hormones

“…Various in vitro studies with liver mince and liver fractions suggest that the hepatic estrogen inactivating system involves cozymase, flavine-adenine-nucleotide and cyto chrome-c. This is in agreement with the findings of DeMeio et al (42) who demonstrated a cyanide-labile system activated by nicotinamide and cozymase effective probably as a dehydrogenating system. Paschkis & Ra koff (43) have presented a detailed review of the recent work of their group.…”

Artificial Estrogens

“…It was suggested that, in part at least, a dehydrogenation mechanism was responsible for inactivation. The inactivating ability of rat liver homogenate is augmented by the addition of diphosphopyridine nucleotide (DPN) and nicotinamide (76, (88), indicating that the postulated dehydrogenation mechanism is in part linked to DPN. Heller (163) and Levy (259) found that cyanide inhibited the ability of liver to inactivate estrogen, but De Meio, Rakoff, Cantarow, and Paschkis (88) found only a limited inhibition by cyanide.…”

The metabolism of [16−14C]oestrone in vitro

“…Levy (1947), using rat-liver homogenates, also showed inhibition by cyanide as well as by azide and carbonmonoxide, and suggested that the cytochrome system was involved. He found that inactivation of oestradiol was greatly reduced under anaerobic conditions, and this was confirmed by De Meio et al (1948), who, however, found very little inhibition of oestrogen-inactivation by cyanide or azide and none by malonate, iodoacetate or fluoride. In all these experiments very little conjugation was observed and the results which were obtained by bioassay methods give no indication about the extent to which the original steroid molecule had been altered.…”

“…Since the original demonstration by Zondek (1934) that liver mince rapidly inactivates oestrogens, many workers have studied the metabolism of oestrone and oestradiol in vitro, employing loss of biological activity to follow hormone-degradation (Heller, 1940;Engel & Rosenberg, 1945;Levy, 1947;De Meio, Rakoff, Cantarow & Paschkis, 1948;Pearlman & De Meio, 1949). Ryan & Engel (1953), by means of countercurrent distribution and fluorometric analysis, found that after incubation of these two natural oestrogens with rat-liver slices over 50 % of the starting material was converted into unknown metabolites.…”