Cytochemical observations with methyl green-pyronine staining showed that the basophilia in the cytoplasm of ascites tumor cells incubated with living hemolytic streptococci at 37°C gradually decreased, and after a 60 minutes treatment cytoplasm vanished and swollen free nuclei remained. After incubation for 90-120 minutes, the staining intensity of desoxyribonucleic acid by Feulgen technique in nuclei more or less decreased. Heat-killed streptococcus, staphylococcus, pneumococcus and Escherichia coli were ineffective in decreasing the cytoplasmic basophilia of the tumor cells. Ribonuclease treatment of tumor cells produced no decrease in cytoplasmic basophilia. The effects of living hemolytic streptococci on ascites tumor cells, however, is not explained by the action of streptolysin S produced in vitro.

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The electron distribution of urethan and its related compounds are obtained using the frontier electron method which has been established by the present authors as one of the molecular orbital methods to discuss chemical reactivity and the results are compared with those of carcinogenesis experiments.
The frontier electron distribution at the carbonyl carbon shows a parallelism with the degree of carcinogenicity of these compounds. This relation leads to the conclusion that the interaction between the carbonyl carbon and the nucleophilic active center in the body might play an important role in the induction of tumor by these compounds and this finding may be an additional support to our postulation that the attack of carcinogen on the nucleophilic center in the body might be involved in the early stage of carcinogenesis.
The sterical resemblance between the urethan and other carcinogens is pointed out and some discussions are made on the active nucleophilic center in the body in connection with the cholinesterase inhibition.
Financial support for this work has been received in part from the Education Ministry of the Japanese Government.

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Changes of pyruvate metabolism during hepatocarcinogenesis by 3'-Me-DAB were investigated, using pyruvate-2-C¹⁴ as a tracer in vivo. The conversion of pyruvate into lactic, succinic, malic, glutamic and aspartic acid and alanine was studied by isolating them by the anion exchange chromatography of Busch et al. The results were as follows:
1) Pyruvate metabolism did not show remarkable changes in the early stage of 2 weeks of 3'-Me-DAB feeding. Marked inhibition was observed in the late stage of 2-3 months, with a gradual advance to a extremely low state in hepatoma. But the difference between precancerous liver and hepatoma was so striking that the existence of some essential difference in the factors controling metabolism was suggested.
2) In explanation of these results, changes of the cellular metabolism, blood supply of the tissue and the cell population were discussed. But, in conclusion, the metabolic change of the cells is thought to be a principal factor.
3) In the carbon tetrachloride poisoned rats, strong inhibition of the pyruvate metabolism was demonstrated in the very early period of 4 hours after the intramuscular injection.
4) Chroline deficient diet caused a considerable inhibition of the pyruvate metabolism in the early stage of 3 days, and this state did not change markedly in the late stage of 30 days.
5) These results were discussed ih regard to the pattern of liver injury by these three substances.

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Multiple pulmonary tumors in mice of dd or C₅₇bl strains were induced by a single injection of suitable quantities of 4-nitroquinoline N-oxide subcutaneously. And mice of strain dd are more susceptible than mice of C₅₇bl to the induction of pulmonary tumors. The observations recorded in this paper indicate that in dd strain mice the lung response occurred in a relatively short period of time, i.e., as early as three months. The nodules of tumors in the lungs per mouse are more numerous when the dose of the agent injected is large than when it was small. Induction of pulmonary tumors in dd mice by the injection of 4-nitroquinoline Noxide is more conclusive than by the injection of 20-methylcholanthrene.

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1. The studies described in this paper were concerned with the relationship between the congo red index and quantitative histology in the rat liver during carcinogenesis by azo dyes, including 3'-Me-DAB, OAT, and M-Orange. Functional test of RES was based on Yasuoka's modified Adler-Reimann method. The histological patterns of the liver were based on the routine examination and Daust's method.
2. The azo dye which had high potency as hepato-carcinogen gave severe disturbances on the functional activity of RES. But it was obscure in the low or noncarcinogenic substances.
3. Histological findings were similar to those reported by various observers. However, the results indicated the exsistence of some relationship between the congo red index and the average percentages of PAS-positive reticulo-endothelial cells.
4. It was concluded that the relationship between the congo red index and the numerical population of various cell types in the rat liver was not remarkable.

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Using the p-dimethylaminoazobenzene feeding at a low level (about 500mg per rat) where there is no liver cancer production, cutaneous applications of 20-methylcholanthrene, but especially of 4-nitroquinoline N-oxide, were shown to bring about the development of liver cancer. Skin painting with 4-nitroquinoline N-oxide or of 20-methylcholanthrene alone failed to produce liver cancer. These results were discussed from the point of view of the summation theory of carcinogenesis, and it was suggested that a small and yet adequate amount of 4-nitrequinoline N-oxide, or its weakly carcinogenic metabolite may reach the liver and complete the submanifestational carcinogenic process already started by p-dimethylaminoazobenzene feeding. The possible "co-carcinogenic" role of inflammatory exudate from the injured skin tissue is not entirely excluded.
It was also demonstrated that 4-nitroquinoline N-oxide is capable of producing skin fibrosarcoma in a high proportion of rats at the site of painting, an unexpected finding in view of the generally accepted fact that the rat skin is insusceptible to tumor producing action of other powerful carcinogens.

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1) When the contents of the intranuclear inclusion produced by a suitable concentration of 4-NQO in tissue culture cells were completely digested with cold PCA, instead of with RNase, it was not stained any more with pyronine and eosin. From this finding the RNP nature of the inclusion was concluded.
2) By the dryice-acetone fixation and subsequent immediate lyophilization, the inclusion was observed as vacuoles which were homogeneously filled with eosinophilic contents. From this result, the morphological constitution of the inclusion surrounded with halo by the ether-alcohol fixation was reconsidered.
3) The fixation and staining with acetogentian-violet were performed with demonstrable results for the specimen treated with 4-NQO. From this finding the nature of the inclusion in relation to the nucleolus was also discussed.

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Toxohormone was separated from normal mouse spleen and from the enlarged spleen of the Friend virus infected mice, three weeks after the inoculation of the virus. Two corresponding fractions from the two sources revealed about the same activity gram for gram, but the Friend spleen yielded each fraction in amount about four times as much as from normal spleen per unit fresh material. The yield ratio was checked against the DNA content of the tissue materials from which the fractions were separated.
These findings demonstrate a greatly increased production of toxohormone by the Friend virus infected spleen and suggest that the huge spleen in this disease may be regarded as approaching malignancy in its peculiar protein metabolism.

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The respiratory and glycolytic activities of Friend virus infected spleen were investigated for the first five weeks after the virus inoculation. The respiratory activity of the virus infected spleen was the same as that of the normal one. Aerobic and anaerobic glycolysis showed a slight increase on the third to fifth week after the virus inoculation. These results were briefly discussed.

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By testing effect in vitro of various homologous tissue extracts under accurately defined experimental conditions it was demonstrated that normal liver contains a potent carcinostatic factor, which other tissues failed to yield. The complete failure of the tumor cells to take, when implanted into susceptible host, was brought about by allowing only 2cc of centrifuged supernatant of mouse liver homogenate, prepared at the rate of 1g of liver in 10cc of normal salt solution, to interact with 2cc ascites equivalent of Ehrlich carcinoma cells at 37°C for 1 hour.
The distinction of this liver factor from the well known oncolytic system of certain heterologous blood sera was pointed out and the possibility of the hepatogenic carcinostatic factor playing a deciding role in the disposal of isolated cancer cells in body fluid and consequently in preventing the formation of metastasis was considered. The importance of homologous carcinostatic system, contrasted to the heterologous one hitherto studied, was especially emphasized.

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The DNA-content of hyperdiploid and hypotetraploid tumor cells of the Ehrlich ascites carcinoma was measured by Feulgen-microspectrophotometry, adopting the two-wave-length method (482mμ and 561mμ) in comparison with that of normal mouse monocytes and splenic cells.
It was found that the DNA-content of nuclei was proportional to the number of chromosomes. The change of the DNA-content during mitosis was measured: it was shown that the mean amounts of DNA of small amount-nuclei at interphase were identical with those of anaphase and telophase nuclei, being just half the metaphasic amount. The amounts of DNA of prophase nuclei correspond to approximately three-quarters of those of metaphase nuclei. Based on the results of measurements of the DNA-content in relation to size of nuclei, the mechanism of DNA synthesis was discussed in connection with cell growth during interphase and prophase. A similar pattern of the correlation between nuclear size and the DNA-content was found to occur in hyperdiploid and hypotetraploid lines.

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