The rôle of period between administration of CCl₄ and feeding of 2, 7-bis-(acetamido)fluorene in the hepatocarcinogenesis of mice was analyzed by using 14 groups of different feeding schedules. Simultaneous treatment with CCl₄ and either continuous or intermittent feeding of 2, 7-bis(acetamido)fluorene increased the average number of hepatic nodules and the incidence of hepatomas, while separation of 8-week feeding of 2, 7-bis(acetamido)fluorene from 8-week treatment with CCl₄ did not accelerate the hepatocarcinogenesis. Alternate treatment with CCl₄ and 2, 7-bis(acetamido)fluorene increased the average number of hepatic nodules but failed to induce hepatomas. Allyl alcohol, which is a hepatotoxic substance as is CCl₄, also failed to accelerate hepatocarcinogenesis of 2, 7-bis(acetamido)fluorene. Concerning the feeding schedule of 2, 7-bis(acetamido)fluorene alone, the intermittent one-week feeding of the carcinogen with 3-week intervals decreased the average number of hepatic nodules as compared with the continuous feeding of the carcinogen.

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Histological studies were made on the nephrotoxic effect of citrinin on the kidneys of rats, with or without previous treatment with the nephrotoxic chemicals, N-(3, 5-dichlorophenyl)succinimide (NDPS) and N-nitrosodimethylamine (DMN). Oral administration of 0.02% or 0.05% citrinin alone caused signs of kidney injury but did not induce kidney tumors. On treatment with DMN alone, 8 of 14 rats (57.1%) developed kidney tumors; two (14.3%) were renal cell tumors, eight (57.1%) embryonal cell tumors, and one (7.1%) hemangioendothelioma. On the other hand, kidney tumors developed in 18 of 19 rats (94.7%) and 13 of 15 rats (86.7%) by the administration of 0.02% and 0.05% citrinin, respectively, after DMN. The tumors in these two groups were diagnosed histologically as renal cell tumors in 18 (94.7%) in group IV and 13 (86.7%) in group III, and as embryonal cell tumors in 14 (73.7%) in group IV and 9 (60.0%) in group III. Thus, in groups treated with citrinin after DMN the incidence of renal cell tumors was much greater and the incidence of embryonal cell tumors slightly greater than in the group treated with DMN alone.
Kidney tumors developed in 4 of 18 rats (22.2%) treated with 0.02% citrinin after NDPS, but treatment with NDPS alone did not induce kidney tumors.
Thus, treatment with citrinin changes the histological type and incidence of kidney tumors in rats induced by DMN. Moreover, this study confirms that citrinin in combination with NDPS can induce kidney tumor in rats, which was reanl cell tumor (adenoma) histologically.

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Experiments were carried out to determine TCD₅₀ (50% tumor control dose) of 3rd generation isotransplants of a C3H mouse mammary carcinoma treated or not treated with Bleomycin. If the antibiotic was injected 30min before a single X-ray dose, TCD₅₀ was reduced. This reduction in TCD₅₀ was independent of Bleomycin dose of more than 15mg/kg, because of the upward-concave nature of Bleomycin dose-cell survival curve. The combined effect, when tested by TCD₅₀ assays, appeared less than additive. This effect was further examined by a series of TD₅₀ assays which revealed that these tumor cells were capable of repairing the potentially lethal damage induced by X-rays and that induced by Bleomycin. It was also found that the potentially lethal damage after combined X-ray and Bleomycin treatments was repaired. These findings indicated that the combined X-ray and Bleomycin treatment resulted in additive effect if the repair of potentially lethal damage in tumor cells were taken into account.

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The in vitro carcinogenicity of sodium nitrite was examined. Addition of a high concentration of sodium nitrite (50mM or 100mM) to mass cultures of newborn hamster cells for 24hr resulted in morphological transformation of the cells. The shortest time required for morphological transformation after this treatment was 21 days. Two of 5 transformed cultures produced progressively growing tumors when injected into young adult hamsters. These tumors were diagnosed as fibrosarcomas. In two control cultures of the same experimental groups, transformed cells appeared 10 weeks or more later than those in treated cultures.
The chromosomes were analysed at several stages of transformation. In the early stages of transformation, chromosome number varied widely from near diploid to tetraploid, but later they became near diploid.
Morphological alteration caused by sodium nitrite was also seen in a shortterm assay, in which hamster embryonic cells (1×10⁴ cells/60mm dish) were treated and morphology was observed 8 days after the treatment.

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Differences in the susceptibilities of the urinary bladder epithelium of Wistar rats, ICR mice, Syrian golden hamsters, and Hartley guinea pigs to three N-nitroso compounds, N-butyl-N-(4-hydroxybutyl)nitrosamine, N-ethyl-N-(4-hydroxybutyl)nitrosamine, and N-butyl-N-(3-carboxypropyl)nitrosamine, were examined histologically. The urinary bladder epithelium of rats was the most susceptible to all three compounds, and especially to N-ethyl-N-(4-hydroxybutyl)-nitrosamine, and bladder cancers developed in all the rats given this compound. The bladder epithelium of mice was less susceptible than that of rats but, although the incidence of cancer was lower than in rats, that of cancer with invasion was higher. Hamsters were far less susceptible than mice, and cancer developed only in 3 of 41 animals given N-ethyl-N-(4-hydroxybutyl)nitrosamine. Guinea pigs were the least susceptible of the 4 species and no tumors were found. Histologically, most of the tumors induced in rats and mice were transtitional cell carcinomas. The incidence of undifferentiated carcinoma was higher in mice than in rats and cellular or structural atypism of the cancer was also greater in mice. Cancers induced in hamsters were all transitional cell carcinomas showing invasion.

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Lentinan, an antitumor polysaccharide from Lentinus edodes, was degraded to seven fractions by treatment with formic acid. The low molecular-weight fractions (I and II) showed no antitumor activity against sarcoma-180 solid-type tumor and the absorption maximum of Congo Red did not shift in their presence in 0.1M sodium hydroxide. The medium molecular-weight fraction III, which required the increase of doses (5 or 10mg/kg) for inhibition of tumor growth, caused a little shift. On the other hand, the absorption maximum of Congo Red shifted largely by the presence of high molecular-weight fractions (IV∼VII) which showed the inhibition ratio of over 95% in a dose of 1mg/kg. Participation of molecular weight in the antitumor activity of polysaccharides which contain (1→3)-β-D-glucan main chain was discussed.

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Experiments were made on induction of cancer of the glandular stomach of rats by a combination of oral administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and insertion of a plastic bead into the gastric lumen. The incidence of cancer with this combination of treatments was greater than by administration of MNNG alone. Fluoroscopic examination showed that barium sulfate remained in the stomach longer when a bead had been inserted into the gastric lumen. This indicates that after insertion of a bead, MNNG must have remained in the stomach longer, and so the period of exposure of the gastric mucosa to the carcinogen must have increased.

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Relationship between the survival time of L-1210-bearing mice and myelosuppression of normal mice after combination chemotherapy was studied. In a variety of combinations of 6-mercaptopurine, 6-thioguanine, and cyclophosphamide, a combination of cyclophosphamide and 6-thioguanine produced the highest increase in life span (ILS) and the highest number of 60-day survivors of all. In contrast, a combination of 6-mercaptopurine and 6-thioguanine showed as low an ILS as did single agents. A combination of 6-mercaptopurine, cyclophosphamide, and 6-thioguanine exhibited the second best effect on survival time of leukemic mice. However, in myelosuppression as measured by changes in the total number of nucleated cells, in the number of hematopoietic colony-forming cell, and in peroxidase level of femoral bone-marrow, the combination of 6-mercaptopurine and 6-thioguanine showed nomore toxicity than the other 2-drug combinations. In addition to these findings, the presence of a striking difference in the cell number in ascitic fluid of leukemic mice among the animals given 6-mercaptopurine and 6-thioguanine, and those given other 2- or 3-drug combinations may suggest that the difference in the antileukemic activity is not due to the difference in the toxicity against the host, but due to the difference in the activity of direct action of combined drugs on leukemic cells in the peritoneal cavity or in other sites.

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The γ-glutamyltransferase (EC 2.3.2.2) (=γ-glutamyltranspeptidase, γ-GTP) activity in hepatoma induced by 3'-methyl-4-(dimethylamino)azobenzene (3'-Me-DAB) was 120-fold higher than that of normal liver and high activity was also found in bovine hepatocellular carcinoma. γ-GTPs from these malignant tissues responded more and showed broader specificity to γ-glutamyl group acceptors than those from normal tissue such as bovine, rat, and mouse liver and bovine kidney. Three species of γ-GTP were isolated from bovine kidney by DEAF-cellulose chromatography, whereas only two species were isolated from bovine hepatocellular carcinoma. The carcinoma lacked the least acidic enzyme species. Appropriate γ-glutamyl group acceptors stimulated more-acidic enzyme species more than less-acidic species in both tissues. The fractions separated from the hepatoma were stimulated much more than those of kidney by γ-glutamyl group acceptor. The enzymes from normal tissues responded similarly to a γ-glutamyl group acceptor irrespective of the difference in their activity. Thus, γ-GTPs of malignant tissues appear to be more versatile for amino acid transport, both qualitatively and quantitatively. In these properties the enzyme of mouse fetal liver which showed the highest activity in the last period of pregnancy resembled the enzymes of malignant rather than normal tissues. The activity of hepatic γ-GTP is not parallel with the rate of cell proliferation during normal development.

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Studies were made on the effect of mucin on the induction of gastric carcinomas by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), with or without sodium chloride, in male Wistar rats. Seven groups of rats were treated as follows: Group 1 was given continuously 50mg MNNG/liter solution to drink and 1ml of saturated sodium chloride once a week and fed on stock diet supplemented with 4% mucin. Group 2 was given 50mg MNNG/liter solution and fed on stock diet supplemented with 4% mucin. Group 3 received 1ml of saturated sodium chloride once a week and 50mg MNNG/liter solution to drink. Group 4 was treated with MNNG only. Group 5 was fed on stock diet supplemented with 4% mucin. Group 6 was given sodium chloride only. Group 7 was untreated. The incidence of gastric cancer in Group 3 was significantly higher than that in Group 4 (P<0.05) or in Group 1 (P<0.05). The difference in the incidence of gastric cancer in Groups 2 and 4, and of intestinal tumors in Groups 1 to 4 were not statistically significant. No malignant tumors were seen in Groups 5, 6, and 7. Thus mucin reduced the high incidence of gastric cancer induced by MNNG and sodium chloride to the level induced by MNNG alone, but it had no effect on the incidence of intestinal tumors. The effect of mucin in preventing destruction of the gastric mucosal barrier by sodium chloride and so reducing the induction of gastric cancer is discussed.

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Studies were made on the induction of tumors in mice by N-butyl-N-nitrosourethan (BNUR), with the following results.
1) Oral administration of 0.04% BNUR in deionized water to CDF₁ mice for 20 weeks produced papillomas in the esophagus and forestomach of all 58 mice, and 24 of the mice had squamous cell carcinoma.
2) No skin tumors developed when 0.1%, 0.5%, or 1% BNUR in acetone was painted on the skin on the back of CDF₁ mice three times a week for 25 weeks.
3) No sarcomas developed within 30 weeks when a dose of 10, 60, or 120mg/ml of BNUR in dimethyl sulfoxide solution was injected subcutaneously into the back of CDF₁ mice, 10 times at 1-week intervals.
4) In the in vitro malignant transformation test, golden hamster lung fibroblast cells did not show malignant transformation during an observation period of 150 days after treatment with BNUR at a final concentration of 30 and 60μg/ml for 24hr.
The carcinogenicity and possible mode of action of BNUR are discussed on the basis of these results.

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Leiomyosarcomas of the small intestine were found in dogs during experimental induction of gastric carcinoma by oral administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Leiomyosarcomas were found most frequently in the duodenum and jejunum, and occasionally in the stomach but never in the ileum, colon, or rectum. The leiomyosarcomas developed in all the dogs given 50μg/ml of MNNG in deionized water to drink but not in dogs fed on porridge food made from standard pellet diet mashed with MNNG at the same concentration in tap water. Intestinal sarcomas developed in 3 months to 5 years after the end of MNNG administration, and frquently metastasized to the liver and/or the peritoneum.

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The antitumor activities of toxoids of Clostridium perfringens, C. novyi, C. septicum, and C. tetani were tested against sarcoma-180 and Ehrlich carcinoma. Among them, C. perfringens toxoid showed a high antitumor activity against the growth of the implanted sarcoma- 180 ascites form. The results of the inhibiting effect of C. perfringens toxoid on Nakahara-Tokuzen-Fukuoka (NTF) reticulum cell sarcoma and methylcholanthrene-induced fibrosarcoma were also described.

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Serum α₁-fetoprotein (AFP) concentration in 5-week-old rats was measured by the radioimmunoassay technique after a single administration of various hepatotoxins. Marked elevation of serum AFP concentrations occurred in rats treated with carbon tetrachloride, thioacetamide, D-galactosamine, allyl alcohol, allyl formate, and ethionine in 4 days of these treatments. The increased production of AFP appeared to be correlated with the induction of liver glucose-6-phosphate dehydrogenase (G-6-PD) among biochemical parameters studied for hepatocellular injuries. However, the difference in time courses of the increase in liver G-6-PD activity and serum AFP level following CCl₄ treatment suggested that the increased production of serum AFP and the induction of G-6-PD in injured liver were caused by closely related but different mechanisms. Pretreatment of CCl₄-injured rats with N, N'-diphenyl-p-phenylenediamine or aminoacetonitrile was effective not only in lowering the increased level of serum AFP and liver G-6-PD but also in preventing liver cell necrosis and steatosis induced by CCl₄. Treatment with a lower dose of thioacetamide resulted in littel elevation of serum AFP and liver G-6-PD with a markedly increased incorporation of ³H-thymidine into liver DNA without any evidence of liver injury. On the other hand, the administration of ethionine, which caused little necrosis of liver cells, produced increase in both serum AFP and liver G-6-PD levels with an only small increase of hepatic DNA synthesis compared to those following thioacetamide as well as CCl₄. These results suggest that the elevation of serum AFP is not directly related to the stimulation of hepatic DNA synthesis. Some additional mechanisms of specific gene amplification for AFP, which is geared to hepatic injury per se, appear to play a major rôle in the increased AFP production in injured liver.

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β-Glucuronidase isozymes in rat tissues were separted by electrophoresis on cellulose acetate membranes. Using this method, β-glucuronidase isozyme patterns were studied in normal rat liver and experimental hepatomas with different growth rates. Changes of isozyme patterns during postnatal development were also studied in rat liver.
Normal rat liver contained six types of β-glucuronidase, numbered I to VI in order of decreasing mobility to the cathode. Type II β-glucuronidase stained most intensely and was detected in all the cells examined. The isozyme patterns of Morris hepatomas, which are slowly growing and not highly deviated, resembled that of normal rat liver but lacked definite Type III β-glucuronidase. Yoshida ascites hepatomas, which are rapidly growing and highly deviated, contained only Type II, but some had Types II, V, and VI β-glucuronidase. Embryonal liver just before birth contained only Type II, but with increase in activity during development after birth, minor bands of Type I and Types III to VI β-glucuronidase appeared successively to complete the adult pattern.

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The rate of ethylation of t-RNA in vivo by L-ethionine[ethyl-l-¹⁴C] markedly lowered in the liver of rats fed a diet containing copper than that of control Incorporation of the labeled compound into the t-RNA of liver by a single injection of L-ethionine[ethyl-l-¹⁴C] was inhibited as much as 50% by the preceding concurrent administration of copper and ethionine added in the diet to rats.
Direct interaction of copper ion with ethylation by ethionine in the specific t-RNA of the rat liver was examined as an important biochemical explanation in molecular level for the inhibitory mechanism of copper on rat liver carcinogenesis by ethionine. Profiles of normal t-RNA on methylated albumin-kieselguhr column chromatography revealed three components for leucine, two of which were found at the tube number 40 to 50 and disappeared in the leucyl t-RNA treated with ethionine. The components for leucine that disappeared were normalized in the liver of rats by the concurrent administration of copper.

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Glucose solution containing wheat straw hemicellulose-B and ovoglycopeptide (W-O-G) has no effect on Ehrlich ascites tumors but completely regresses Ehrlich solid tumors. The carcinostatic effect of this mixture is lost or decreases markedly whenever one of the three substances is lacking. For this glucose solution to possess a carcinostatic effect it is necessary that this hemicellulose-B of W-O-G is the hydrolysate of the extract with 20% NaOH from the residue obtained after extraction of wheat straw with 10% NaOH.

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The rôle of the kidneys in hepatic carcinogenesis was studied in inbred Buffalo strain male rats ingesting 0.04% N-4-(4'-flourobiphenyl)acetamide in the diet. Experimental groups were made up of male rats with both kidneys intact and male rats with the left kidney removed. The incidence of carcinomas of the liver and the number of rats with large carcinomas, multiple carcinomas, poorly differentiated and undifferentiated carcinomas, and metastases were greater in rats with a uninephrectomy. Apparently the animals with one kidney removed were unable to secrete the metabolites of N-4-(4'-fluorobiphenyl)acetamide as readily as the rats with both kidneys.

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Tumor cells were coupled with fluorecent dansyl group in aqueous medium by dansyl chloride-cycloheptaamylose complex (CDC) without destruction of the cells. C57BL/6 mice and Donryu rats pretreated respectively with dansylated EL4 leukemia cells and with dansylated Yoshida sarcoma cells acquired transplantation immunity to the corresponding tumor cells. Serum and spleen cells obtained from EL4 immune mice showed cytotoxicity to EL4 cells but not to other allogeneic leukemia cells. Hapten-specific cytotoxicity of immune serum and spleen cells was not observed in the present immune system.

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The antitumor activity of berberine, berberrubine, and their derivatives against sarcoma-180 ascites was determined by the total packed cell volume method. Berberine and tetrahydroberberine derivatives had no antitumor activity, but berberrubine (9-demethylberberine) and the ester derivatives of berberrubine had a strong antitumor activity. ED₉₀ of berberrubine, its acetate and benzoate, were 15, 23, and 44mg/kg, respectively. The therapeutic indices (LD₁₀/ED₉₀ by the present method) of these compounds were as follows: Berberrubine hydrochloride, 6.7∼9.4; 9-acetyl-9-demethylberberine (9-acetylberberrubine) chloride, 7.6∼8.7; 9-benzoyl-9-demethylberberine (9-benzoylberberrubine) chloride, 3.4∼4.9.

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Cytotoxic cytidine analog, cyclocytidine, and antitumor immunopotentiator, lentinan, offered an opportunity of testing the effect of combined modality of cytocidal agent and immunopotentiator on spontaneous mammary tumors of mice. Lentinan did not improve the therapeutic effect of cyclocytidine; it only prevented the early toxic death of mice due to cyclocytidine in one strain of mice but not in the other strain.

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