In conclusion, some attempts to detect steroid-mediated hydrogen transfers in androgen-sensitive tissues of the male genital tract may be worthy of mention, in that they stress some of the difficulties encountered in this type of experimentation. Homogenates of the prostate gland and seminal vesicle, from which nuclei and large cytoplasmic particles have been removed, degrade pyridine nucleotides very rapidly (141). Enzymatic rupture occurs at many positions in these molecules, and the rate of dephosphorylation of TPN(H) to DPN(H) is particularly fast. With such tissue extracts, and even with some more purified preparations obtained by further fractionation with ammonium sulfate, it is difficult to maintain adequate pyridine nucleotide levels for the assay of hydrogen transfers between TPN and DPN. The effects of testosterone on the reoxidation of reduced pyridine nucleotides by extramitochondrial fractions of seminal vesicle (141) appear to be related to an inhibition of a microsomal DPNH oxidase system in this tissue (140).

VI. Model Systems For Hydrogen Transport By Phenolic Estrogens

The placental enzyme which catalyzes estrogen-dependent hydrogen transfers between pyridine nucleotides reacts only with steroids bearing a 8-hydroxyl or ketone function at position 17. Since 1933, when Cook, Dodds and Hewett (21) discovered that l-keto3-1:2:3:4 tetrahydrophenanthrene elicits estrus in ovariectomized rats, a wide variety of nonsteroidal substances have been found to exert estrogenic action. Vaginal cornification can be induced by such chemically dissimilar substances as triphenylethylene; hydroxylated stilbenes like diethylstilbestrol and hexestrol; doisynolic and allenolic acids; isoflavones such as genistein; dibenzanthracene diols; and even simple monophenols such as 4-tert-amylphenol (26).

Most of the synthetic estrogens are devoid of either secondary alcohol or ketone groups. Consequently powerful estrogens such as diethylstilbestrol do not mediate transhydrogenations catalyzed by the placental 17β-hydroxysteroid dehydrogenase (117). The statements of Villee and his collaborators (38, 126) that diethylstilbestrol has a weak but definite activating effect upon the placental steroid-sensitive transhydrogenase are not supported by their own data, which simply indicate that high concentrations of this substance, and related stilbenes, can compete with the action of estradiol-17β in this enzyme system. The idea that synthetic estrogens may undergo metabolic transformations into steroid-like structures has been entertained by many authors, and for the majority of these substances there is no experimental evidence for or against this possibility. But studies with C14 labeled diethylstilbestrol (41), and with tritium-labeled hexestrol (27), have not disclosed biochemical changes of this nature.

Since the finding of Gordan and Elliott (32) that diethylstilbestrol, as well as certain steroids, depresses glucose oxidation in brain tissue, the inhibition of various mammalian oxidizing enzyme systems by natural and synthetic estrogens in vitro has been reported from a number of laboratories. Some of the enzymes of the tricarboxylic acid cycle and of the terminal electron transport pathways of mitochondria appear to be particularly sensitive in this respect. Thus, the succinic (17, 87) and malic (87, 97) oxidase systems of many mammalian tissues are inhibited by diethylstilbestrol and related phenols. Some of these substances primarily inhibit cytochrome oxidase, whereas the action of others may be on the primary dehydrogenase, or on some intermediate steps in these multienzyme sequences (17). Oxidative phosphorylation in liver (100) and prostate (137) mitochondria is uncoupled by diethylstilbestrol, a property also shared by progesterone (132). In all of these studies, inhibitory activity could not be correlated with biological activity. Moreover, the concentrations of synthetic estrogens required to depress the action of these enzyme systems more than 50% usually exceeded 5 X 10-5M. Thus it is difficult to attribute physiological significance to these inhibitory actions of synthetic estrogens. However, the mechanism of the inhibition of certain oxidizing enzymes by such hydrophobic phenols is obscure and merits further investigation.

Hochster and Quastel (45, 46) found that, with manganese dioxide as a terminal hydrogen acceptor, diethylstilbestrol increased the rate of anaerobic oxidation of ethanol, lactate, a-glycerophosphate, and hexosediphosphate by yeast acetone powder extracts, and of D-alanine by a crude preparation of kidney D-amino acid oxidase. In the yeast-MnO2 system, the carrier action of methylene blue for the oxidation of all these substrates was inhibited by diethylstilbestrol, which also depressed the oxidation of ethanol and a-glycerophosphate (but not that of lactate or hexosediphosphate) when ferri-cyanide was present. Similarly, diethylstilbestrol interfered with the carrier function of cytochrome c when lactate was used as substrate. Hochster and Quastel suggested that diethylstilbestrol was converted into its quinone form by the action of MnO2, and postulated that the quinol-quinone system could either transport hydrogen as such, or interfere with the carrier action of other electron acceptors. The chemical synthesis of what was believed to be diethylstilbestrol quinone had been reported previously by von Euler and Adler (130), although a crystalline product could not be obtained. Hochster and Quastel (46) reported that this quinone, like the parent diethylstilbestrol, inhibited the aerobic oxidation of lactate catalyzed by a yeast acetone powder extract plus a brain homogenate, and presumed that these substances competed with the terminal electron transport system. However, the concentrations of diethylstilbestrol used in the experiments of Hochster and Quastel were so high (2.3 X 10-8 M) that it is difficult to envisage their observations as being physiologically meaningful.

The extremely low concentrations of steroidal estrogens required to mediate transhydrogenations catalyzed by the placental 17β-hydroxysteroid dehydrogenase prompted a search for other enzymatic systems for hydrogen transport by low levels of both natural and synthetic estrogens. Two such model systems have been studied in some detail (138, 139). Many simple nonestrogenic phenols transport hydrogen in both types of reaction, but much information concerning the reactivity of estrogenic substances has been gained. The first system is catalyzed by some plant phenolases and, at least in the case of estradiol-17β, implicates an o-diphenolic derivative of the estrogen as a hydrogen carrier. The second model reaction is catalyzed by certain peroxidases in the absence of added hydrogen peroxide, and appears to involve phenoxy radical derivatives as hydrogen transporting intermediates. The peroxidase model system may have some bearing on the metabolic function of a peroxidase in mammalian uterus, which is particularly sensitive to estrogenic hormones in vivo.

1. Phenolase-Catalyzed Oxidations

Phenolases catalyze two types of transformation of phenols in the presence of molecular oxygen: (a) hydroxylation of monophenols to o-diphenols, and (b) oxidation of o-diphenols to o-quinones (85, 86). Otto Warburg (71, 72) discovered that these enzymes are copper proteins, and showed that reduced pyridine nucleotides were among the hydrogen donors which are oxidized by phenolases when incubated with catalytic quantities of appropriate o-di-phenolic substrates, as follows:

If phenolases hydroxylate phenolic estrogens in an ortho position, it can be envisaged that low levels of the products could transport hydrogen in this manner. This possibility was examined in the light of the following facts:

(a) Westerfeld (134) originally showed that plant phenolases "inactivate" steroidal estrogens. According to Graubard and Pincus (34, 35), the inactivation of estrone and estradiol-17β by mushroom copper oxidases is an oxidative reaction, during the course of which three to four atoms of oxygen are consumed per mole of estrogen added. Colored reaction products were obtained, but they were not characterized. However, the recent experiments of Jellinck (60) showed that mushroom phenolase(s) almost certainly attacks estrone only in ring A, and that the degradation products differ, at least in part, from those obtained by the incubation of estrone with liver slices. Zondek and Sklow (144) claimed that potatoes contain an estrogen-inactivating enzyme which is not identical with phenolase. But this suggestion was shown to be in error by the careful experiments of Bergstrom, Theorell, and Westman (10), who demonstrated that the estrogen-inactivating and phenolase activities of potato extracts paralleled one another during quite extensive purification.