1. Homogenates of normal and tumor tissues were respectively fractionated into lipid and residue (protein) fractions and these fractions were examined for lipid peroxide forming activity. It was found that whereas neither the lipid nor residue fraction alone showed apperciable activity, the combination of both fractions invariably showed a high peroxide value, at least in the case of normal tissues. 2. Lipid which is responsible for the peroxide formation was found to be phospholipid. 3. When the residue fraction was dialyzed, the peroxide forming activity was no longer observed even after the addition of an active lipid fraction, suggesting the persence of a dialyzable cofactor in the residue. It was therefore concluded that for the production of lipid peroxide a phospholipid and a cofactor are essential together with zinc. 4. Iron alone could activate the lipid peroxide formation in whole homogenates or lipid fractions, although zinc has been found to be the only metal which can restore the activity in EDTA-treated homogenates. 5. A comparison of the activity of peroxide formation was made in various reciprocal combinations (tumor fractions×fractions of normal liver). Having confirmed that capability of peroxide formation is nearly all lost in tumor tissues, it was further found that the peroxide value was greatly decreased when tumor lipid was added to the residue of normal liver. 6. Reduced activity in tumor tissues with respect to peroxide formation is not due to the presence of antioxidants. 7. Suggestions were put forward that as the result of neoplasia some qualitative changes in phospholipid occur and at the same time zinc becomes firmly bound to cell structures and no longer available for the reaction, these changes leading to an overall inhibition of the lipid peroxide formation in tumor tissues. 8. The general impression was obtained that the tumor tissue is different from normal in the nature of phospholipid constituents, and that the modification or the lack of certain phospholipids constitute an important characterization of the tumor tissue.
1) Cancerous gastric juices were more labile polarographically than those of the anacidic and control samples. 2) Comparing the Pfw of each specimen of gastric juice aspirated at intervals of 20 minutes after insulin-injection, a transient lowering of Pfw of a specimen taken at the first 20 minutes was notable in gastric cancer. 3) From the chemical determination, the content of nitrogen, tyrosine, DPA-reactive substance, hexosamine and hexuronic acid was found to be in the descending order: Cancerous, anacidic, and acidic (control) samples. 4) As the result of statistical inquiry of the analytical data, positive correlations were found to exist between the DPA-reactive substance and the hexosamine content. No correlation was noticed between the combined content of tyrosinenitrogen and that of DPA-hexosamine, so that the substances increased in cancerous gastric juice may be devided into two different groups, i.e. the nitrogentyrosine group and the DPA-hexosamine group. 5) From the result of acidic control, a physiologically active substance in Pfw (typical double wave) was noticed in Fr. VIII, which consisted electrophoretically of a single peak B1, and into which the grandular Mp has been fractionated by Glass and Boyd. 6) Contrary to the above finding, the polarographically active substances in the cancerous specimens were observed in Fr. VI and VII, which consisted electrophoretically phoretically of the ill-difined peaks, B2, B3 and B4. The increases in the content of DPA-reactive substance and of hexosamine seemed to be related to these peaks appeared in Fr. VI and Fr. VII. 7) The anacidic specimen was placed between the acidic control and the cancerous specimen in view of the lability of Pfw and the characteristics of chemical composition, but it resembled, in the nature, more closely to the cancerous specimen rather than the control specimen. These facts described above may indicate that there is the functional precancerous stage of gastric secretion, on which further investigation will be made.
In comparison to the histamine refractory anacidic specimen of several gastric disorders with acidic control and with carcinomatous specimen, the following conclusions were obtained by electrophoretical investigation in both native gastric juice and in fractionated specimens according to the method as reported by Glass and Boyd. 1. The electrophoretic pattern in native gastric juice of histamine refractory anacidics was placed between that of healthy control and carcinomatous gastric juice. That is to say, the electrophoretic pattern of healthy acidic control shows B1 and B4 peak clearly, as compared with B2 or B3 peak. The pattern of carcinomatous gastric juice differs from the control pattern in two points, showing that an increase of B2 and B3 peak and diminution of B1 peak were present. On the other hand, in the histamine refractory anacidics, increase of B2 and B3 peak is seen as in the case of carcinomatous specimen, whereas B1 peak is clearly observed as in the case of the control. 2. In the fractionated specimen, FVIII (MP) is closely connected with B1 peak (at pH 8.6) or a- and b- peak (at pH 4.5) electrophoretically. FVII (Ms) is connected with B4 peak (at pH 8.6). B2 and B3 peak, which increased in both histamine refractory anacidics and cancerous specimen, seemed to be connected with FVI and FVII. It is likely that the increase in FV, VI and VII corresponds with the above pathological patterns. Moreover, it was ascertained that the both fractions of MP (FVIII) and Ms (FVII) in pathological gastric juice consisted of heterogenous components electrophoretically. 3. A considerable part of the constituents in gastric juice with the exception of MP is contained in FV, which is a supernatant of acetone-soluble fraction. And these constituents are transferred largely into FV in the case of pathological gastric juices, i. e., histamine refractory anacidics and carcinomatous gastric juices. Accordingly, it is postulated that acetone-soluble fraction can not be neglected in the pathological gastric juices.
1. Polarographical waves and biochemical contents of orcinol reactive, diphenylamine reactive substance, glucosamine, glucuronic acid, tyrosine and of nitrogen were measured on fractionated specimens by means of continuous electrophoresis with regards to three groups of gastric juices, the healthy acidic control, the gastric carcinoma and the histamine refractory anacidic sample. 2. Electrophoretic patterns obtained by continuous electrophoresis agree with that of strip paper electrophoresis. Thus, it was ascertained that the above chemical analyses were performed on electrophoretically homogenous specimens. 3. In the acidic control, the highest polarographical filtrate wave and the highest contents of orcinol chromogen, diphenylamine chromogen, and of tyrosine were observed in B1 peak with the fastest mobility. Hexosamine content was highest in B4, and was also found to exist in the area between B1 and B2. The contents of hexuronic acid and nitrogen were noticed in B4 with slow mobility. 4. In both histamine refractory anacidics and carcinomatous gastric juice, it was found that the above chemical contents observed in B1 were transferred into B2 or B3. And it was of interest to note that the histamine refractory anacidic sample was placed between the acidic control and the carcinomatous sample with respect to their electrophoretic patterns and characteristic behaviours of chemical constituents. Thus the clarification of functional precancerous stage may be expected by investigating chemical constituents, in detail, which are related to B2 and B3.
1) The activity of histidase in the liver of rats fed 4-dimethylaminoazobenzene (DAB) was studied rather qualitatively by means of paper electrophoresis. The activity was represented in a pattern in consideration of the length of migration of enzyme on the paper. 2). Normal rat liver showed one prominent peak on the activity pattern at the electromigrated location, which reached nearly to an end of the zone of tissue protein. Regenerating liver showed also a similar pattern to that of normal liver. 3) The liver of DAB fed rats in their early experimental days, the cirrhotic liver and hepatoma of prolonged DAB fed rats showed low activity pattern, especially low in hepatoma. 4) Qualitative changes of histidase have not been detected in the course of hepatocarcinogenesis, although the activity diminished keeping step with the progress of pathological changes of the liver.
1) Acid and alkaline phosphatase in the homogenate of rats fed 4-dimethylaminoazobenzene (DAB) were investigated by means of paper electrophoresis and the activity was represented in a pattern, details of which have been described. 2) Pattern of acid phosphatase in normal liver showed two prominent peaks at the initial point and at migrated zone of protein respectively. Alkaline phosphatase showed a low maximum at the initial point and a very low curve situated at the migrated part. 3) Patterns of both phosphatases in cirrhotic liver were similar to those of normal liver, but there was a higher peak at the initial point, especially in the case of alkaline phosphatase. 4) In case of hepatoma, pattern of alkaline phosphatase showed a peak of high level at the initial point, higher than that of normal liver and even of cirrhotic liver. The peak of acid phosphatase at this site was lower than that of normal and cirrhotic liver. 5) Patterns of both phosphatases in the livers of DAB fed rats in their fourth week were alike to those of hepatoma. 6) Patterns of alkaline phosphatase in sera of DAB fed rats showed non-detectable differences from that of normal rat sera. A marked peak of bell-shaped curve was observed in α2-globulin fraction and lower one at the starting point.
1) Ribonucleic acid (RNA) desaminase in the liver of rats fed 4-dimethylaminoazobenzene (DAB) was studied by means of paper electrophoresis. The activity was expressed by the amount of liberated ammonia and the extent of electromigration of enzyme on the paper was also represented on the activity pattern. 2) Whole homogenate of normal liver, the pathological liver including hepatoma, and the liver of DAB fed rats in their fourth week of experiment showed the similar pattern, in the shape of plateau extending to whole protein zone, although having a slight undulation especially at the peak of the starting line. 3) In the pattern of RNA desaminase in acid and alkaline media, no characteristic difference was observed among the above mentioned hepatic tissues. 4) The pattern of the supernatant covered nearly all the moving part of that of the whole homogenate. The pattern of the sediment coincided with that of the slow moving part of the whole homogenate.
The present communication deals with studies of the urinary excretions of tryptophan, kynurenine and anthranilic acid in tumor bearing rats or mice before and after the administration of tryptophan. In the rats with cirrhotic or cancerous livers urinary excretion of tryptophan and its metabolites were less than normal. And the urinary excretion of tryptophan in tumor-bearing mice was of subnormal proportion. However, the kynurenine level in the urine was high in these mice.
The response of the transmissible venereal tumor of dogs to x-ray irradiation has been studied. Tumors of eight female dogs, in the vaginal wall adjacent to the labia, disappeared completely following radiation therapy given in six exposures of 200, 300 or 400r over a period of three weeks (1200 to 2400r total dose). From the results obtained it is concluded that the transmissible venereal tumor of the clog is sensitive to irradiation. Histological studies revealed transient suppression of mitosis, cellular and nuclear enlargement and mitotic aberrations in the earlier stages of treatment. In the advanced stages of tumor regression, nuclear disintegration, infiltration with granulocytes, lymphoid cells, histiocytes and plasma cells followed by proliferation of fibrous tissue were observed. Both clinical and hematological observations indicate that the irradiation therapy was well tolerated.
In the rats which received the transplantation of the ascites tumor, MTK-sarcoma III, the changes in contents of glycogen, RNA and DNA in the liver cells were investigated by means of a series of cytochemical and microspectrophotometric methods. Gradual glycogen depletion was consistently observed from experiment to experiment in the liver cells of rats bearing tumor transplants. Every rat which died of tumor contained a very little amount of glycogen in their liver cells. The rate of the glycogen depletion of tumor-bearing animals exceedingly surpassed that of starved rats. It has remained unsolved in the present study whether the observed depletion of liver glycogen occurred in rats as a direct response to the enormous requirement of energy by tumor cells, or whether tumor transplants attacked hormonal glands of the host which control the glycogen storage in the liver. Whatever the causes may be, it is very likely that the glycogen depletion observed in the tumor-bearing rats may be attributable to the tumor transplants in hosts. Contrary to the glycogen depletion, RNA (basophilia digestable by RNase) increased gradually with the prolonged existence of the tumor transplants. The increase in amount of RNA was observed both in the cytoplasm and in the nucleus. The thickening of the nuclear membrane and the hyperactivity of the nucleolus observed in the tumor animals seem to indicate the active participation of the nucleus in association with the accelerated production of RNA in the liver cells. An increase of net DNA content (Feulgen-Schiff complex) took place in connection with an increase of the nuclear volume in liver cells of tumor-bearing animals during the period of the most active proliferation of the tumor. Its significance was discussed in relation to the DNA content of the nucleus. Increased mitotic activity and dilatation of the liver sinusoids were also observed in livers of the rats bearing tumor transplants.
CYTOLOGICAL STUDIES OF TUMORS
XXIV. AN AZO-DYE INDUCED ASCITES HEPATOMA OF THE RAT, WITH SPECIAL REMARKS ON TRANSITIONAL CHANGES OF NEOPLASTIC CHARACTERS IN THE COURSE OF SERIAL TRANSFERS
The present study deals with several transitional changes occurring in the neoplastic property of an azo-dye induced ascites hepatoma of the rat since its development. With the accretion of transplant generations, the hepatoma islands showed a gradual change in general structure. There occur also transitions in transplantability and life-span of the tumor-bearing animals during the course of serial tranfers. In earlier transfer generations, the chromosome number of tumor cells of this hepatoma fluctuated within a very wide range, without showing any distinct modal values. With the increase of transfer generations, the chromosome number showed a rather narrow range of variation with the occurrence of distinct modal values at 69-71. It can be can be concluded on the basis of the present findings that the constancy of the stem-line, while it is very pronounced, is not necessarily fully stable, and that under certain conditions the tumors have undergone numerical and structural changes in their stemline-chromosomes during serial transfers. Apparently such chromosomal changes will cause the transition of the genotype of the tumor which will in turn produce the shifts in the property of the tumor.