Immunoelectrophoretic method and Ouchterlony's gel double-diffusion method have been applied for the antigen analysis of the sera from patients bearing malignant tumors. The materials investigated were obtained from about 320 cases of various malignant tumors and from about 250 of normal or non-tumorous cases for control.
Immunoelectrophoretic patterns of the sera of tumor patients against absorbed specific antisera showed the presence of several abnormal precipitin lines which could not be demonstrated in normal sera.
In the sera of cancer patients, most of the abnormal antigens were demonstrated in the α₂- and β₁-globulin fractions. The distribution of these antigens differed in individual cases and showed no specific changes correlated to the histopathological classification and primary sources of tumors. It is, however, worthy of notice that these antigens increased in parallel with the growth of tumors and disappeared or markedly decreased after the extirpation of tumor tissues.
Almost all sera of myeloma, leukemia, and malignant lymphomas formed one characteristic line in the β₂-globulin region and they could be differentiated from the patterns of cancerous materials.
It may be assumed that the immunological demonstration of abnormal constituents in sera is useful for the detection or identification of many malignant tumors.

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Various aminoazo dyes (100μmoles/rat) were administered to rats with a stomach tube and aminoazo dyes were extracted from the liver 15 hours later, fractionated by column chromatography and assayed. The following aminoazo dyes were used: 4'-F-DAB, 4-F-MAB, 4'-F-AB, 3'-Me-DAB, 3'-Me-MAB, 3'-Me-AB, 2', 3-diMe-DAB, 2', 3-diMe-MAB, 2', 3-diMe-AB, DAB, MAB, AB, 4-ethylmethylaminoazobenzene, and 4-monoethylaminoazobenzene.
It was confirmed that the secondary and tertiary aminoazo dyes were easily convertible into each other in vivo and also they gave corresponding completely N-demethylated primary aminoazo dye, but the primary aminoazo dyes, such as 2', 3-diMe-AB (OAT), 4'-F-AB, or 3'-Me-AB which had been speculated to be N-methylated in vivo to become carcinogenic, were proved not to be N-methylated. However, these primary aminoazo dyes including AB gave several unknown metabolic products which were different from any one of the N-methylated derivatives and seemed to be of complex nature. Some discussions were made concerning the steric effect of a substitution at 3-position upon N-methylation as well as the amount of dyes recovered from the liver with respect to the reductive cleavage rate of the azo bond reported in the previous paper.

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Four unknown dye components, Fractions 0 to 4, were discovered in liver of mice or rats administered with the so-called o-aminoazotoluene (OAT). Isolation and purification procedures as well as chemical and physical properties of these dyes are described. Among these, Fr. 0 and Fr. 4 were obtained in a crystalline form and were found to have twice the molecular weight of OAT. Chemical structure of Fr. 0 was determined and identified as 4, 4'-bis (o-tolylazo)-2, 2'-dimethylazobenzene (BTDA) which was synthesized from OAT by oxidative condensation with manganese dioxide. Although exact chemical structure of Fractions 1, 3, and 4 was not clarified yet, they seemed to belong to aminoazo dyes. From the studies on their reduction products, they may have been derived from hydrazo form of BTDA or other condensation product by semidine or diphenyline rearrangement.
Significance of the presence of Fr. 0 or other related compounds in the liver was discussed with special reference to the N-hydroxylative metabolism of aminoazo dyes.

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To study the influence of dietary protein on the development of hepatoma induced by 4-dimethylaminoazobenzene (DAB), five types of 0.06% DAB diet of different composition were given to male adult rats of Sprague-Dawley strain. These diet contained (1) 20% casein, (2) 20% peptide mixture derived from casein, (3) 20% casein-pattern amino acid mixture, (4) 20% amino acid mixture of casein pattern except tryptophan which was about one-half of casein, and (5) 20% amino acid mixture deficient in tryptophan, threonine, and lysine. The incidence of hepatoma was significantly higher (p<0.05) in groups fed on diets (4) and (5), which were deficient in one or more essential amino acids, than in those given diet (1), (2), or (3).
There were no significant differences in the incidence of hepatoma among the groups given (1), (2), and (3) which contained casein, casein pattern peptide, and amino acid mixture. Regarding the incidence of fibrosis and cirrhosis, there was no difference among the groups studied. The present study revealed the importance of quality of dietary protein in the inhibition of DAB-induced hepatoma.

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Relationship between the growth of Ehrlich ascites carcinoma and changes in the hyaluronic acid content in the ascitic fluid was investigated. The concentration of hyaluronic acid in ascitic fluid was maintained at about 40mg% during the active development of tumor cells and then increased abruptly in the later stage.
When tumor-bearing mice were treated with D-galactosamine, the total amount and concentration of hyaluronic acid in the ascitic fluid on the 12th day after tumor inoculation decreased significantly. All the anticancer agents tested, except Nitromin, caused an increase in the hyaluronic acid concentration.
To elucidate these changes further, hexosamine synthesis in tumor cells was examined and the strongest activity was observed in the earlier stage of tumor development. The hexosamine synthesis in tumor cells was not affected either in vitro or in vivo by amino-sugars or anticancer agents other than Nitromin.
Morphology and histochemistry of tumor cells were also studied and it was suggested that the elevation of hyaluronic acid concentration in the ascitic fluid might be related to the breakdown of tumor cells.

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In view of the disturbance of iron metabolism in tumor-bearing animals, possible potentiation of the carcinostatic activity of anticancer agents by iron compounds against Ehrlich ascites carcinoma in mice was examined.
It was found that the carcinostatic activity of the alkylating agents used was potentiated by ferrous glucuronate or ferrous fumarate, which are both inactive when given alone. Ferric glucuronate did not have the same effect. Carcinostatic activity of purine antagonists tested was not enhanced by administration with these iron compounds.

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The activity of iron-protoporphyrin chelating enzyme in the liver of tumorbearing mice was almost the same as that of normal mice and was found to be very low in tumor cells. Of the anticancer agents tested in vitro, 2, 5-bis(ethyleneimino)-1, 4-benzoquinone and 6-mercaptopurine inhibited this enzyme activity in normal mice.
Daily decrease in the levels of aconitase, total ascorbic acid, and non-hemin iron in the liver was observed after tumor inoculation, while the liver protoporphyrin level first increased and then decreased. During this period, a gradual increase in concentration of non-hemin iron in the tumor cells was observed. Administration of Thio-TEPA to tumor bearers restored these metabolic disturbances to normal in parallel with its therapeutic effect. Ferrous glucuronate or ferric glucuronate failed to produce this effect, but they maintained a high level of the PIII iron fraction in the liver of tumor bearers. Administration of Thio-TEPA with ferrous glucuronate, however, rapidly restored these altered metabolism of tumor bearers to normal and greatly increased the level of liver ferritin iron(PII). Combination therapy of Thio-TEPA with ferric glucuronate failed to maintain the high level of liver PII iron, although the aconitase, protoporphyrin, and total ascorbic acid contents were restored almost to normal. In this case, high level of the PIII iron fraction was observed. When 6-mercaptopurine was given to tumor bearers, the lowered levels of aconitase and non-hemin iron in tumor bearers were depressed further and the subsequent return to normal was delayed. Further, the high protoporphyrin level in tumor bearers persisted during 6-mercaptopurine therapy. Administration of 6-mercaptopurine with ferrous glucuronate to tumor bearers caused no appreciable elevation of the liver PII iron level, while the aconitase, protoporphyrin, and total ascorbic acid contents were restored to normal.
Treatment with ascorbic acid, which slightly decreased the mean survival time of tumor-bearing mice, caused a marked decrease in the liver PII iron level.
Significance of these results in relation to those in the previous report is discussed.

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A spontaneous mammary carcinoma in a high-leukemic AKR strain of mice was studied both in vivo and in vitro, and an attempt was made to elucidate the mechanism of sarcomatous transformation. The tumor, originally a type B adenocarcinoma, transformed into a sarcomatous tumor during serial transplantation in isologous mice. Cell strains were established in vitro from transplants before and after the sarcomatous change. The cultured cells were composed of epithelial and fibroblast-like cells and their intermediate forms. It has been shown that (1) the carcinoma cells assumed a spindle shape in loosely growing areas, (2) in the mixed carcinosarcomatous stage, the epithelial tumor cells often merged with the stromal cells, indicating a morphological transition from polygonal to elongated cells, and that (3) the behavior and morphology of the cultured cell strains were similar, although they were derived from histologically different transplants. These findings suggest that the sarcomatous transformation in a serially transplanted AKR mammary tumor resulted from a morphological alteration of the carcinoma cells.

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A comparison was made between antigenic structure of two strains of rat ascites hepatoma, AH-49 and AH-41B. Precipitin reaction of rabbit antisera against tumor proteins extracted from these cancer cells indicated that AH-41B cells possessed a specific antigenic protein which was not found in AH-49 cells.

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Subcutaneous injection (1mg/week for 20 weeks) of 6-carboxy-4-nitroquinoline 1-oxide, a water-soluble derivative of potent carcinogen 4-nitroquinoline 1-oxide induced fibrosarcomas in 3 out of 13 rats, about 300 days after the final administration.

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A series of 4-alkyltriazeno-imidazole (or -triazole)-5-carboxamides were tested for their growth-inhibitory effect on Ehrlich tumor. Of the compounds tested, 4-dimethyltri-azenoimidazole-5-carboxamide and 4-dipropyltriazeno-imidazole (or -traizole)-5-carboxamide were found to be the most effective inhibitor of solid tumor. Except for 4-diazoimidazole-5-carboxamide, none of the compounds tested had any effect on the ascites form.

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