In order to clarify the mechanism and process of the development of free-cell sublines of the ascites hepatoma AH-130, double inoculation experiments of freecell sublines, AH-130F(G) and AH-130F(N), and their original strain AH-130 were designed. According to the observation, time necessary for conversion into free-cell form was relatively long and it was not always related to the ratio of tumor cell number of free-cell subline and original strain in the inoculum. Further, there was a much difference in the time necessary for conversion between AH-130F(G) and AH-130F(N), when they were inoculated respectively in the same host with AH-130, although both were derived from the same original strain AH-130. All cases of the double inoculation did not always convert into the free-cell form and some did not show any shift, so far as successive transplantation was performed for more than 200 days. In comparison of the time necessary for conversion between successive transplantation with metastasized tumor cells in the liver and that with tumor ascites following the double inoculation of AH-130F(G) and AH-130, the former achieved the conversion more rapidly than the latter.

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Macroscopical and histological features were examined on 171 cases of intramucosal and submucosal cancers of the stomach and following results were obtained.
1) The cancerous foci were classified macroscopically into elevated (Type I), superficial (Type II), and excavated (Type III) types according to the nature of the affected mucosa. The cases consisted of 21 of Type I, 34 of Type II, and 116 of Type III.
2) Type II cancers were sub-classified into slightly elevated (II-a), flat (II-b), and eroded (II-c) types according to changes in the mucosal surfaces. The 34 cases of Type II consisted of 2 of II-a, 5 of II-b, and 27 of II-c types.
3) The cancerous tissues of the mucosa were classified histologically into welldifferentiated (Type A), less-differentiated (Type B), and poorly differentiated (Type C) adenocarcinoma. Macroscopical types described above were compared with this histological pattern. All cancers, which were classified into I, II-a, and II-b, had pictures of A or A to B type histology but no such histological inclination was observed in Type II-c and III.
4) The histological patterns of the intramucosal cancer were closely correlated with grade of intestinal metaplasia in the cancer-bearing pyloric mucosa, regardless of macroscopical types of the cancer.

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Using an isolated perfused liver, failure for induction of tryptophan pyrrolase (TP) in the liver of rats bearing Walker carcinoma-256 was studied, together with the inhibitory effect of toxohormone on the TP induction.
When the isolated liver of a normal rat was perfused with normal blood containing hydrocortisone, TP increased 4-fold after 6 hours of perfusion, while, in the isolated liver of rats bearing Walker carcinoma-256, whose tumor weighed about 10% of the body weight, TP was not significantly increased by the circulation of hydrocortisone.
With the perfusion medium containing toxohormone the circulation of a normal liver resulted in a marked reduction of TP.

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A chromosome study was carried out in effusion cells derived from a patient with neuroblastoma in in vivo and in vitro conditions. The majority of in vivo and in vitro cells showed a normal chromosome complement, probably of normal cell origin from the host.
Cells with abnormal karyotypes were observed in effusion cells in a small proportion. The chromosomal aberrations were recognized as non-random loss or gain of certain chromosomes, occurring rather frequently in group 13-15, and less frequently in groups 16-18 and 6-X-12. Structurally abnormal chromosomes such as dicentrics, acentrics, ring, and new unusual elements were also observed in some effusion cells, particularly in those in vitro. These changes might have been, at least in part, caused by X-ray treatment given to the patient.

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Liver tumors were induced in mice of A/Jax strain after a single, subcutaneous injection of 0.4 or 0.7mg of o-aminoazotoluene within 24 hours after birth. Among 26 treated males, 12 males (46.2%) developed liver tumor during the observation period of 15 months, whereas, among 27 females, only 5 females (18.5%) developed tumors, a significant sex difference being observed. This is quite contrary to the incidence of liver tumors in the male and female of A/Jax strain mice that received monthly injections of the same azo dye starting at young adult age. Partial hepatectomy performed at 6 months of age augmented liver tumor development after neonatal injection of o-aminoazotoluene. In both males and females, hepatic lesions demonstrated by histologic observation occurred as early as 24 hours after the injection, and reached the maximum on the 20th day. These hepatic lesions were repaired almost completely within a few weeks. However, the liver of mice exposed to o-aminoazotoluene at birth still showed histological alteration, i.e., variation of cell size, which might persist throughout the observation period. It seemed that such hepatic alteration was more prominent in the male than in the female. The possible significance of hepatic changes induced by o-aminoazotoluene injection on sex difference in the incidence of liver tumors is briefly discussed.
It was also observed that neonatal injection of o-aminoazotoluene resulted in a fairly high incidence of pulmonary adenomas, and a slight, but probably significant, increase in leukemia incidence.

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The manner in which 20-methylcholanthrene causes increase in hepatic dimethylaminoazobenzene-demethylase activity of rats was studied.
The effect of methylcholanthrene is not mediated through hormonal glands, for comparable increases in demethylase activity following administration of methylcholanthrene were observed in intact, adrenalectomized, ovariectomized, and hypophysectomized rats. When hypothalamic lesions were made in rats, the basal level of demethylase decreased slightly and the response of this enzyme to methylcholanthrene was significantly, though not completely, impaired. On the contrary, glutamine synthetase, a microsomal enzyme not induced by methylcholanthrene treatment, was not affected by hypothalamic lesions of rats. Thalamic lesions of rats had no influence on the response of hepatic demethylase to methylcholanthrene. These results suggest that the hypothalamus is of importance for the maximal increase in demethylase activity after methylcholanthrene treatment.

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Based on an idea that ortho-hydroxyl derivatives of carcinogenic aminoazo dyes might be responsible for the dye-protein binding, reaction of 3-hydroxy-4-aminoazobenzene (3-HO-AB) and 3-hydroxy-4-monomethylaminoazobenzene (3-HO-MAB) with various amino acids and amines was investigated. Studies indicated that lysine and histidine gave reaction products upon heating them in alkaline solutions with the dyes. Under tha same conditions, tyrosine gave a dye-bound product to a much less extent, while the other amino acids did not. Some chemical and physico-chemical parameters such as absorption spectra, reactions with Folin reagent, Ninhydrin and Gibbs reagent, electrophoretic mobility at various pHs, and chromatographic behavior of the reaction products were compared with those of the natural polar dyes prepared from dimethylaminoazobenzene (DAB)-fed rat liver. Several alkylamines were also investigated. Among them the primary and secondary alkylamines gave reaction products, but tertiary amines and ammonia did not. Specific reaction mechanisms were presented for the primary amines including lysine and for the secondary amines including histidine.
It was confirmed that the natural polar dyes do not possess a phenolic group in the dye moiety, at least in the free state. This fact necessitates the presence of a phenolic group in the amino acid moiety or the presence of more than two amino groups in the dye moiety.

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Rates of reductive cleavage of the azo group in various aminoazo dyes as one of the competitive (or non-carcinogenic) metabolic pathways of the dyes were studied in vitro system using a rat liver homogenate fortified with NADPH-generating system in nitrogen atmosphere. The azo reduction rates observed for these dyes were compared with their carcinogenic potencies, π-electron densities at azo nitrogen atoms (α and β), and acid dissociation constants, pKa', of the azonium ions. The results indicated that a single parameter such as azo reduction rate could not be correlated with the carcinogenic potency. A linear relationship was observed between π-electron density at β-azo nitrogen and pKa'. Discussions were made on the biochemical approach to understand the carcinogenic activity of aminoazo dyes from metabolism studies.

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Penetration of benzo[a]pyrene and other aromatic hydrocarbons into the esophageal mucosa after application into the esophagus of mouse was examined by fluorescence microscopy. When benzo[a]pyrene was dissolved in aqueous solutions of ethanol and administered, the epithelium of the esophagus showed a strong purplish blue or occasionally whitish blue fluorescence, However, the epithelium hardly fluoresced when the same hydrocarbon was dissolved in olive oil and administered in entirely the same way. This does not necessarily mean, however, that benzo[a]pyrene in oil can never penetrate into the esophageal epithelium because a moderately strong fluorescence was observed in the epithelium after a highly concentrated solution of benzo[a]pyrene in Tricaprylin was similarly administered. It was also noted that benz[a]anthracene and fluoranthene dissolved in ethanol solution or aqueous caffeine solution can enter the epithelium of the esophagus.

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1) Tumor-inducing effect of two nitroso compounds was tested on the mice. Three strains of mice, ddN, ICR, and C3H/AHe were divided into groups and fed with N-nitrosodimethylamine or N-nitrosodiethylamine, the former mixed in the basal diet and the latter added to the drinking water. Concentration of the dimethylamine was in 50, 100, and 200p.p.m., and the length of administration was either 5 or 10 months. The animals were given basic diet and observed until death.
2) Following kinds of tumors were induced by this treatment: adenomas of the liver in 16 cases, carcinomas of the liver in 6 cases, hemangioendothelial sarcomas of the liver in 14 cases, adenomas of the lung in 63 cases, adenocarcinomas of the lung in 19 cases, papillary cystadenomas of the kidney in 5 cases, malignant tumors of the kidney in 2 cases, seminomas in 2 cases, leukemias in 3 cases, forestomach papillomas in 7 cases, and one hemangioendothelial sarcoma of the subcutis.
3) There were certain differences among the strains and between the two compounds.
4) The results were compared with those already reported by other investigators and the mechanism involved is discussed.

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An idea of histochemical differential demonstration of the lactic dehydrogenase isozymes has been introduced and its procedures have been described.
Inhibition of the activities of each lactic dehydrogenase isozyme subunit at the substrate, coenzyme, and enzyme levels by various kinds of chemicals and by physical conditions was investigated. Nicotinamide-hypoxanthine dinucleotide, an NAD analog, guanidine hydrochloride, urea, and heat were found to be useful for this purpose. The basic data and histochemical results are given, and the mechanisms involved are discussed.
1) The activity of H-type LDH is histochemically demonstrable in tissues by applying NHXD, an NAD analog, in place of NAD, or by adding guanidine hydrochloride or urea in the standard incubation medium.
2) The activity of M-type LDH is also histochemically demonstrable in tissue by pretreating the frozen, air-dried tissue sections through hot water at 60°.
3) These methods were verified by a biochemical analysis using agar-gel electrophoresis conducted in parallel.
4) All the possibilities for developing histochemical techniques for demonstrating the LDH isozyme subunits were discussed.

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Antitumor activities of Tubercidin, Toyocamycin, and their derivatives were investigated using NF-sarcoma in mice. It was found that amino group at 4-position and ribofuranosyl group at 7-position were necessary, at least, for the antitumor activities of these molecules.

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