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Continued administration of several hepatocarcinogens led to an increase in the concentration of glutathione (GSH) in the livers of intact, but not of hypophysectomized or adrenalectomized rats. The concentration of GSH remained high untill the development of hyperplastic nodules. Subsequently, the concentration of GSH dropped to the normal level or below. A single dose of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) produced an increase of GSH which, within a certain range, depended upon the amount of the carcinogen. In well differentiated, slowly growing hepatomas, the concentration of GSH approached the level in normal adult rat liver. On the other hand, in nondifferentiated and rapidly growing hepatomas, GSH was only 30-40% of that in normal liver. The activity of gamma-glutamyl transpeptidase (GTase) increased within 24-48 hours after a single large dose of 3'-Me-DAB. Continued feeding of 3'-Me-DAB led to an exponential increase of GTase. During hepatocarcinogenesis, the level of GTase activity corresponded to the degree and size of pathologic changes produced in rat liver. Chloramphenicol partially inhibited the increase of GTase induced by 2-acetylaminofluorene. Pretreatment with 3-methylcholanthrene partially inhibited the increase of GTase that had been induced by a single dose of 3'-Me-DAB. Puromycin partially inhibited the increase of GTase induced by several doses of dimethylnitrosamine. These observations indicated a close connection between the activation of GTase and chemical carcinogenesis in rat liver. Measurements of GTase activity in 12 Morris hepatomas supported this conclusion; their GTase levels were greatly elevated compared with that in normal adult rat liver.

1. 1. Using the dual label method, relative rates of protein degradation were examined in liver fractions of rats after prolonged feeding or the carcinogenic 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) and the non-carcinogenic isomer, 2-methyl-4-dimethylaminoazobenzene (2-Me-DAB). 2. 2. After 50 – 63 days of continual feeding, both azodyes cause a decrease in the rates of protein degradation of all liver fractions studied. Within this time period, the effect is more pronounced in the case of 2-Me-DAB. 3. 3. The accumulation of rat liver mitochondria as a consequence of prolonged 2-Me-DAB administration as observed by earlier workers is attributed to decreased degradation rather than to increased biogenesis. 4. 4. Mitochondrial protein subunits, in the presence of sodium dodecylsulfate, can be separated by Sepharose gel chromatography into two distinct fractions: a fairly homogenous high mol. wt fraction characterized by a low turnover and low degree of 2-Me-DAB binding, and a heterogeneous lower mol. wt fraction characterized by high turnover and higher degree of 2-Me-DAB binding. 5. 5. The pronounced effect of the noncarcinogenic 2-Me-DAB on rat liver may provide a convenient and useful system in which the mechanism of protein degradation in the mammalian cell can be studied.

The concentration and total amount of DNA in the livers of SD rats fed 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) gradually increased and reached a maximum in developing tumors. In SD rats fed 3'-Me-DAB plus disulfiram (DSF), the concentration of DNA was higher than in controls, but it soon became stabilzed and the total amount of DNA in the liver did not differ substantially from that in rats fed DSF alone. In rats given 3'-Me-DAB, neoplastic nodules and liver carcinomas appeared after 3 months, but in those fed both compounds these formations were absent even after 6 months. The activity of gamma-glutamyltransferase (GT-ase), a marker of chemically induced carcinogenesis in rat liver, gradually increased to extremely high levels in tumors even after 75 days when the diet of the animals was changed to a normal one. In rats fed 3'-Me-DAB plus DSF, GT-ase activity increased for the greater part of 80 days, gradually leveled off around the 100th day, and returned to almost normal levels when the rats were given a normal diet after 100 days. We concluded that DSF 1) did not interfere with 3'-Me-DAB-induced proliferation of preneoplastic cells and the increase in GT-ase associated with this reversible adaptation to the influx of 3'-Me-DAB; and 2) inhibited malignant transformation and, consequently, prevented the formation and proliferation of neoplastic cells and the increase in constitutive GT-ase related to neoplasia.

On the Correlation between Metabolic and Structural Changes During Carcinogenesis in Rat Liver

Under special conditions dried yeast preparations can be obtained which show a typical Pasteur effect with a normal quotient (MQ). mol CO 2 fermentation suppressed by oxidation mol O 2 consumed of 1 to 2.5. Quickly dried bakers' yeast without further treatment was used in the first place. The Pasteur effect is strictly reversible. Moreover, it is nearly completely inhibited by para-nitrophenol (1·10−3M) and less completely by 2,4-dinitrophenol and 3,5-dinitro-o-cresol. While almost no phosphate is esterified anaerobically, some phosphorylation occurs in oxygen, and this latter phosphorylation is then completely inhibited by para-nitrophenol. Because such a dried bakers' yeast still contains 50% cells able to grow, the yeast was treated with ultrasonic vibration for 60 to 80 mins. Although this destroys some ATP-ase, the fermentation is still fairly constant for 60 to 80 min in the presence of low concentrations of phosphate. All observations can be repeated with the vibrated yeast with the same results. This yeast is now permeable to cofactors (cozymase, ATP, etc.) and the fermentation rate is tripled by addition of optimal concentrations of these factors. Another yeast type, Spiegelman's yeast K, gives the same results without ultrasonic treatment. It contains only 2% of living cells. It shows a specific activation by cozymase which has been treated for some minutes with carbonate (ph 10.5). This activation is weaker in oxygen. This leads to a real or apparent increase of the MQ up to 4.

Intracellular Localization of Carcinogen and its Relationship to the Mechanism of Carcinogenesis in Rat Liver

Ribonucleotide composition of rat adrenal tissue after ACTH stimulation was studied by ion-exchange chromatography. One hour after hormonal administration, a pronounced shift of ribonucleotides to a high-energy level of phosphorylation was indicated.

1. Homogenized tissue mixed with Pyronin Y can be separated by differential centrifugation into red-stained nucleic acid-rich elements—nuclei and chromidia —and unstained nucleic acid-poor mitochondria. Pyronin can thus be useful in preparations of pure mitochondrial suspensions. 2. Chromidia thus obtained correspond to basophilic elements of cytoplasm observed in histological preparations. By electron microscopy, RNA content and sedimentation in gravitational field, they appear as a heterogenous population of granules of various sizes. Their RNA content (basophilic) appears to increase in proportion to decrease in their size and sedimentability. 3. Mitochondria of mouse liver and of certain mouse tumors appear to have a constant proportion of RNA to nitrogen (basophilic quotient). In rat pancreas a higher value was found. 4. Liver, pancreas and tumor tissue show completely different patterns in distribution of basophilia. Tumor tissue is characterized by scarcity of large granules (mitochondria and heavy chromidia), reduction in the amount of small chromidia (microsomes) and predominance of basophilia in non-sedimented smallest nucleoprotein granules. Tumors are thus distinguished from such tissues as pancreas in which mitochondria are also scarce but heavy and light chromidia abundant. 5. A considerable succinoxidase activity was invariably found in large chromidia. This activity was inhibited by incubation with crystalline ribonuclease.

The theory of knowledge is justified only by its results. Each of its statements is essentially an hypothesis, valuable in so far as its practical purpose is fulfilled. Consequently, the same speculative characteristics common to all natural sciences, must be attributed also to the theory of knowledge. All speculative statements constructed for practical purposes when reduced to simple fundamental formulae have no meaning whatever. In the bare form they cannot be proved. Despite this we assume that with the help of such formulae the language may be formed which finally corresponds to the language of nature itself. Logic, on the other hand, is only a play with unproved axioms to form semantic formulae. The combination of axioms is governed by rules as is every other sort of game. Instead of pawns or stones the symbols appear representing the objects of our interest in nature. The rules governing such a game are based upon one condition only: that in every combination the element independent of us may be clearly recognized and further used for other practical purposes in various other combinations. That this basic function can be performed is based on our inner organization rather than on some kind of analogy between the laws of nature and the rules according to which our expressions are formed. Anyway, we are not in the position to know in advance the rules of the process of knowledge any more than we are able to know the mechanism of breathing simply because we breathe. The semantic formulae and the rules of logic do not represent the pathway of the process itself but only its result. It is our inner organization which is responsible for the correspondence finally found when the object of our interest is reconstructed in knowledge. Thus the elementary processes of knowledge have to be analyzed with the tools which are the product of knowledge itself. Similarly to breathing lungs providing oxygen to other organs and to the lungs themselves, the information about the elementary processes of knowledge may be provided only by the mechanism about whose functions nothing is known in advance. Even were we once able to perceive all details of the physiological process of knowledge we shall still be bound to express them in formulae which are products of the same physiological function. So it is necessary that we interpret the theory of knowledge in the light of its tools and any effort to pass over this physiological circle cannot make sense. Nevertheless the corresponding agreement between nature and our interpretation of nature's laws brings forth a conviction of a deep fundamental harmony between both of them. The theory of knowledge ranks with the special branches of natural science, each of which builds up the statements formally in some way of different character. To analyze them is one of the purposes of the theory of knowledge. Of course, it is useless to subdivide the complex of natural sciences, except for practical purposes, since any wall between them can be only a temporary one. 253

The growth rate of transplantable adrenocortical tumors in male LAF 1 mice was found to be inhibited in hypophysectomized and in adrenalectomized animals. Castration and sex hormones did not affect the growth of these tumors. In intact mice a striking inhibition of the growth rate of adrenocortical tumors was brought about by the administration of ACTH. Evidence was obtained that ACTH inhibited growth by a direct action on the tumor cells. When expressed in terms of units of DNA, the biochemical and cytochemical effects on adrenocortical tumor cells appeared qualitatively similar to the effects of this hormone on the normal adrenal cortex. A single injection of ACTH stimulated the activity of glucose-6-phosphate dehydrogenase. Repeated administration of ACTH produced an increased oxygen uptake by sliced tissue and a moderate accumulation of RNA in the microsomal fraction. Water extracts of acetone powdered, ACTH-treated, adrenocortical tumors had lower dCMP-deaminase activity than extracts from untreated tumors. When expressed in terms of units of DNA, the homogenates of ACTH-treated adrenocortical tumors had a higher succinoxidase activity and the sliced tissue had a lower rate of anaerobic glycolysis than did corresponding samples of untreated adrenocortical tumors. FACTEURS ENDOCRINIENS DU DEVELOPPEMENT DES TUMEURS TRANSPLANTABLES DE LA CORTICOSURRENALE I. INHIBITION DE LA CROISSANCE PAR LA CORTICOTROPHINE (ACTH) Il a ete constate que la vitesse de croissance des tumeurs transplantables de la corticosurrenale chez des souris LAF 1 mǎles etait ralentie chez les animaux soumis a l'ablation de l'hypophyse et des surrenales. La castration et les hormones sexuelles n'avaient aucun effet sur la croissance de ces tumeurs. Chez les souris intactes, l'administration d'ACTH a provoque un ralentissement frappant de la croissance des tumeurs corticosurrenaliennes. On a recueilli des donnees montrant que l'ACTH exerce cette action de facon directe sur les cellules tumorales. Exprimes en fonction de l'unite d'ADN, les effets biochimiques et cytochimiques sur les cellules tumorales sont apparus qualitativement semblables aux effets de cette hormone sur le cortex surrenalien normal. Une injection unique d'ACTH a stimule l'activite de la glucose-6-phosphate deshydrogenase. L'administration repetee d'ACTH a provoque une augmentation de l'absorption d'oxygene par le tissu decoupe et une accumulation moderee d'ARN dans les microsomes. Les extraits aqueux de tumeurs corticosurrenaliennes traitees par l'ACTH et „poudrees” a l'acetone avaient une plus faible activite de la dCMP-desaminase que les extraits de tumeurs non traitees. Exprimes en fonction de l'unite d'ADN, les homogenats de tumeurs traitees par l'ACTH avaient une plus grande activite de la succinoxydase et le tissu decoupe un plus faible taux de glycolyse anaerobie que les specimens non traites.

THE carcinogenic azodye 3′-methyl,4-dimethyl aminoazobenzene induces gross tumours in livers of rats fed a basal diet poor in riboflavin1. The whole process, in Sprague–Dawley rats, takes approximately 200 days. Structural damage to liver tissue may be detected histologically during the first weeks of carcinogen feeding2. Intracellular changes, manifested by the gradual depletion of mitochondria and ergastoplasm, which result in respiratory damage to the cell, can be detected by differential centrifugation weeks and even months before onset of malignancy3; yet all these changes are long-range effects. Even though the continual administration of this carcinogen brings about respiratory damage to the cell, the carcinogen is not a respiratory inhibitor4,5. It can be shown that the specific respiratory activity (activity per mgm. protein nitrogen) with sodium pyruvate as substrate remains normal for mitochondria during feeding with this carcinogen although the tissue is full of protein-bound azodye5. In short, the immediate chemical effect of this carcinogen, apart from its binding to one or more proteins not yet functionally characterized6, is still obscure.

Evidence is presented to show that Morris Hepatoma 5123 A differs strikingly from homologous normal tissue in its metabolic and structural aspects. The succinoxidase activity of Hepatoma 5123 A is only 40% that of liver, while anaerobic glycolysis, although relatively low, is nevertheless some 100% higher than in normal liver. The mitochondrial content of Hepatoma 5123 A is about 40% that of the normal liver cell. The amount of material in the microsomal fraction is reduced in the hepatoma and there is a pronounced shift in the distribution of RNA in favor of the RNA in the nuclear and supernatant fractions. Hepatoma 5123 A shares all of these characteristics with other known tumors. In distinction to rapidly growing tumor and proliferating tissue, Hepatoma 5123 A has about the same or higher dCMP-ase activity as normal liver, while dCMP-deaminase is about 20 times as high as in normal rat liver. In addition to the quantitative depletion of mitochondria, Hepatoma 5123 A shows also a depletion in the flavin and cytochrome c content of the mitochondrial soluble matrix. In contrast to normal mitochondria, in which the administration of 3,3',5-tri-iodothyronine led to a an increase several times the normal value, in the activity of α-glycerophosphate dehydrogenase, the level of this enzyme in tumor mitochondria remained unchanged following the administration of triiodothyronine. In contrast to normal liver, Hepatoma 5123 A has shown in all subcellular fractions the presence of polarographically active, non-protein SH-compounds. In normal rat liver these compounds appear only in the mitochondrial fraction and their level is much lower than in Hepatoma 5123 A. It is suggested that the Warburg type of metabolism in Hepatoma 5123 A may be a manifestation of structural and functional insufficiency of preformed cytoplasmic elements.

A factor which promotes the inactivation of Ehrlich ascites TMP kinase (EC 2.7.4.9), TDP kinase (EC 2.7.4.6) and dCMP deaminase (EC 3.5.4.12) in vitro was partially purified from rat-liver microsomes. The factor is heat-labile and has a large molecular weight. During preincubation of this factor with TMP kinase and TDP kinase, TMP kinase is inactivated to a greater degree than TDP kinase. The inactivation can be partially prevented by the presence of TMP, thymidine or mercaptoethanol during the preincubation. The microsomal factor has no detectable phosphatase or proteolytic activity.

Nachweis, dass experimentelle Hepatome in der Ratte eine hohe Glutathionase-Aktivitat bewirken. Dasselbe gilt auch fur die embryonale, nicht aber fur die adulte Rattenleber.

ACTIDIONE (cyloheximide) inhibits protein synthesis in yeasts, certain protozoa and mammalian cell lines1–3. It is a toxic compound for some mammalian species, for example, rats; however, in these cases, hydrocortisone acts as an antidote against such poisoning4. We recently reported5 that actidione in rats stimulates the adrenal cortex by way of the anterior pituitary. The stress reaction protected rats against actidione while hypo-physectomy or adrenalectomy made them more sensitive to actidione poisoning5. Furthermore, mice (which are normally highly resistant to actidione poisoning4,5) became sensitive to actidione after adrenalectomy5.

BARIUM carbonate, used as an adsorbent for stable (platelet-free), oxalated, horse plasma (shaking with 10 mgm./ml. for 30 min. at 0° C.) removes prothrombin with an undialysable, thermolabile substance, destroyed by heating to 60° C. for 30 min. This substance, apparently an albumin, when added to non-activated plasma (without addition of brain thromboplastin), acts as an inhibitor of plasma coagulation.

The relationship of SH- and SS-groups to growth processes has been the subject of much research1. An increase in the concentration of SH-groups was usually associated with the mechanism of mitosis2. Since the observations of Hammett3 and Voegtlin4 the relatively high rate of cell division in tumours has been reported to be connected with greater amounts of glutathione in malignant tissue. Measurements by Greenstein of the glutathione content of spontaneous and transplantable hepatomas indicated values some 10 per cent and 24 per cent lower than those of normal liver on a wet-weight basis5.