One of the considerations leading to these experiments was that hydrogen atoms at critical sites of the steroid molecule are of decisive importance in the promotion of cell growth by compounds exerting androgenic or estrogenic effects. Another was the higher incidence of hepatic disease, cirrhosis and cancer especially, in males than in females, in the rat and in man. What biochemical differences between male and female liver exist to explain sex-linked predominance of hepatic diseases?

The liver is in a special category as a target organ in its responses to hormonal modifications of the milieu interieur. The concentrations of three soluble hepatic dehydrogenases requiring pyridine nucleotides as hydrogen acceptors were influenced considerably by endocrine changes. Not all organs were similarly responsive; for example, hypophysectomy caused a considerable decrease in the concentrations of 6-phosphogluconic dehydrogenase (6-PGD) and glucose-6-phosphate dehydrogenase (G-6-PD) in liver but had little effect on their levels in kidney, spleen, heart, or lung. Not all hepatic enzymes were influenced by hormonal modifications; often the concentrations of acid and of alkaline phosphatases were uninfluenced by hormonal effects which profoundly modified levels of dehydrogenase activities.

It was learned in the experiments (69) that multiple endocrine factors influence the levels of hepatic dehydrogenases; of these hormones, thyroxine and steroids both in the C18 and in the C19 series have high significance. The multiplicity of factors accounts for the seemingly complex observations.

Glock and McLean (Biochem. J., 61:390, 1955) discovered that thyroxine influenced the level of 6-PGD and G-6-PD in liver, an observation confirmed by us. In the present work, it was observed that steroids in the androstane and estrane series exert powerful and, under stated conditions, opposing effects on the levels of G-6-PD and 6-PGD in liver.

In normal rat after birth each of three hepatic dehydrogenases, lactic dehydrogenase (LAD), 6-PGD, and G-6-PD increased in an individually characteristic pattern from the relatively low levels of late fetal life. The concentration of LAD had risen considerably at age 3 days; the concentration of isocitric dehydrogenase has been found to increase in a similar manner. In the case of LAD the highest values observed in the present experiments were found at age 15 days; but at age 20 days, the level characteristic of adult rats was found. It would appear that this rise of LAD in early life was determined primarily by metabolic causes.

After birth, the hepatic concentration of 6-PGD remained at the low level of fetal liver until age 26 days; then in female rats, but not in males, a gradual increase of concentration occurred until high values were found at age 57 days. Likewise in female rats, the hepatic concentration of G-6-PD remained at the low level of late fetal life until age 50 days, when it rose considerably; no rise of G-6-PD occurred in the liver of untreated males. The developmental patterns of 6-PGD and G-6-PD appear to be primarily influenced by hormones.

The level of LAD in liver was profoundly modified by the presence or absence of thyroid hormones-by administration of L-thyroxine or by thyroidectomy. Changes in the steroid status of the animal exerted no highly significant influence on the hepatic concentration of LAD in the present experiments.

The experiments demonstrated that two hormones (estradiol-17β and L-thyroxine) can increase the level of 6-PGD in liver. The concentration of 6-PGD in liver of adult female rats was reduced by ovariectomy. Gonadec-tomy in the male was not followed by a decline in the hepatic level of 6-PGD because the testis of the rat secretes only small amounts of estrogens. Hypophysectomy profoundly reduced the hepatic level of 6-PGD in both sexes.

The level of 6-PGD in liver was considerably augmented by the administration of estradiol-17β to ovariectomized rats. It is significant that this rise did not occur prompdy; there was an interval of 12 days after commencing the injections before high values of 6-PGD were found in liver of every rat. Elevated levels of 6-PGD induced by estradiol-17β were prevented by the simultaneous injection of dihydrotestosterone and by 3α-hydroxysteroids and the degree of inhibition had a stoichiometric relation to the quantity of the androstane derivative which was administered. Estradiol-17β did not induce an elevation of the level of 6-PGD in thyroidectomized or hypophysectomized rats. But the level of 6-PGD rose in livers of rats, deprived of thyroid or pituitary after the administration of L-thyroxine alone-and to especially high levels when estradiol-17β was injected together with L-thyroxine. Thyroxine-induced elevation of 6-PGD in liver was rapid-significandy elevated values were observed after three days in contrast to the delay following the administration of estradiol-17β.

Three hormonal factors (69) were found to augment the concentration of G-6-PD in liver: (a) testosterone, (b) estradiol-17β, and (c) thyroxine; testosterone was augmentative only when administered with estradiol-17β. The concentration of G-6-PD in liver was higher in adult females than in males; the level was reduced somewhat by ovariectomy. But orchiectomy did not lower the hepatic level of G-6-PD in the male.

The hepatic levels of G-6-PD of adult females were much depressed by (a) administration of dihydrotestosterone or (b) hypophysectomy, or (c) thyroidectomy. Remarkably, each of these procedures was equivalent in its effectiveness. Moreover, the concentration of G-6-PD in adult male liver was of the same order of magnitude as that found in hypophysectomized females.

In intact males, low hepatic values of G-6-PD are explained by the presence of effective amounts of testosterone; in castrate males, the low values are due to the absence of effective quantities of phenolic estrogens or thyroxine.

Estradiol-17 did not cause an increase in the concentration of G-6-PD in ovariectomized females, but it was highly effective in this regard in castrate males. In spayed females, and in males as well, the hepatic level of G-6-PD was markedly increased when testosterone was administered together with estradiol-17β. But testosterone administered alone depressed the level of G-6-PD in intact females. Why, in gonadectomized rats, does estradiol-17β elevate the hepatic level of G-6-PD in males and possess no such effect in females? It would appear that the castrate male possesses a synergistic factor, akin to testosterone, which is deficient in spayed female rats; indeed, castrate males grow more rapidly after gonadectomy than spayed females do, but the reason for this disparity between the sexes has not been elucidated. L-Thyroxine always induced a rise in the level of G-6-PD in the livers of rats of both sexes.

There exists a basal level of concentration of the three dehydrogenases in liver which must be essential for life of the cell. It was possible to effect an increase in the level of 6-PGD without promoting an increase above the minimal level of G-6-PD; the opposite was not observed-increased values of G-6-PD were always accompanied by elevated levels of 6-PGD as well.

We see that each of three hepatic dehydrogenases has peculiarities of response (69) to modifications of the endocrine status of the rat, and gonadal steroids and thyroid hormones are of significance in determining the hepatic levels of these enzymes. But thyroid hormones and steroids are not equivalent in effectiveness. Thyroxine is pre-eminent in determining the levels in the liver of LAD, G-6-PD, and 6-PGD as shown by the following evidence: (a) Thyroidectomy reduced the concentration of each dehydrogenase to a basal constitutive level, whereas modification of the steroid status fails to modify gready the level of LAD. (b) The concentration of each dehydrogenase was elevated following the administration of L-thyroxine, whereas in ovariectomized females estradiol-17β increased the concentration of only one of the dehydrogenases (6-PGD) under discussion, and the estrogen was ineffective in this regard in the absence of thyroid hormones.

Are there two physiologic mechanisms, respectively, steroid- or thyroid-dependent, for increasing the levels of 6-PGD and G-6-PD, or do the great effects of the steroids on the concentration of hepatic enzymes result solely from modifications of the rate of secretion of thyroid hormones which steroids might induce as a secondary effect? The evidence favors two separate mechanisms in liver for controlling the hepatic concentration of dehydrogenases: for (a) thyroxine raised the levels of 6-PGD, G-6-PD, and LAD in spayed female rats, whereas estradiol-17β increased the concentration of 6-PGD alone; and (b), dihydrotestosterone, administered concurrently, blocked the capacity of estradiol-17β to increase the concentration of 6-PGD but did not depress the augmented levels of dehydrogenases induced by thyroxine.

In sum, modification of the endocrine milieu interieur by administration or withdrawal of hormones considerably influenced the concentration in liver of lactic dehydrogenase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconic dehydrogenase. Hormones effective in this regard are thyroxine, estradiol-17β, dihydrotestosterone, and 3 α-hydroxysteroids in the androstane series. These hormonal effects are both organ-selective and, in liver, enzyme-selective.

Thyroxine and steroids are not equivalent in their influence on the three hepatic dehydrogenases. Thyroxine was pre-eminent in this regard among hormones investigated in the present experiments.

Each of the pyridine nucleotide-linked dehydrogenases in liver had an individually characteristic response to the administration of various hormones which was reflected in its hepatic level. The concentration in liver of lactic dehydrogenase was profoundly raised or lowered, respectively, by the administration of thyroxine or by thyroidectomy. Injection of steroids had only a small influence on the level of this enzyme.

The levels of glucose-6-phosphate dehydrogenase and of 6-phosphogluconic dehydrogenase were considerably influenced by injection of thyroxine, estradiol-17β, or testosterone. Sex steroids did not increase the levels of these enzymes in hypophysectomized or thyroidectomized rats unless thyroxine was administered concurrendy.

Under designated conditions, it was found that the concentration of 6-phosphogluconic dehydrogenase was increased by hormonal methods which did not elevate the concentration of glucose-6-phosphate dehydrogenase. But high levels of glucose-6-phosphate dehydrogenase were not achieved without a considerable elevation of 6-phosphogluconic dehydrogenase as well.