A neutral form of glutathione S-transferase (GST-P), which has a subunit of 26, 000 (or 21, 500) daltons and charge isomers with isoelectric points of 6.7 (major), 6.3 and 6.0, was purified from rat placenta cytosol. Immunohistochemical staining using anti-GST-P rabbit antibody demonstrated that GST-P, which is hardly detectable in normal liver, is markedly increased and localized in pre-neoplastic hepatic lesions such as hyperplastic nodules, indicating that GST-P could be a useful marker proteins for preneoplasia in chemical hepatocarcinogenesis.

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When a neutral solution of N-nitrosomorpholine, N-nitrosopyrrolidine, or N-nitrosopiperidine was irradiated with near-ultraviolet light, the solution became mutagenic to Salmonella typhimurium TA100 in the absence of metabolic activation. The formation of the active compounds required the presence of phosphate or its esters such as adenosine 5'-triphosphate during the irradiation.

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The mutagenic compounds 2-amino-6-methyldipyrido [1, 2-a:3', 2'-d] imidazole (Glu-P-1) and 2-aminodipyrido [1, 2-a:3', 2'-d] imidazole (Glu-P-2), which were isolated from a glutamic acid pyrolysate and are potent carcinogens in the liver and brown adipose tissue of mice, were found to be multipotent carcinogens in rats. These compounds were each given to F344 rats of both sexes at a concentration of 500ppm in pellet diet for up to 24 months. Glu-P-1 induced tumors in the colon, small intestine, liver, Zymbal gland, clitoral gland and brain. Glu-P-2 produced tumors in the same sites at slightly lower incidence. The multipotent carcinogenicities of Glu-P-1 and Glu-P-2 in rats and mice suggest that heterocyclic amines present in cooked food may be important in the development of human cancer.

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The effects of flavonoids, antioxidants and related compounds on 12-O-tetradeca-noylphorbol-13-acetate (TPA)-caused epidermal ornithine decarboxylase (ODC) induction, DNA synthesis and skin tumor promotion, and on epidermal lipoxygenase activity, were investigated using CD-1 mice. Morin, fisetin, kaempferol and n-propyl gallate potently inhibited epidermal lipoxygenase activity, and esculetin, butylated hydroxyanisole (BHA), α-naphthol and 2, 3-dihydroxynaphthalene (2, 3-DHNA) moderately inhibited it. α-Tocopherol, (+) catechin, (-) epicatechin and butylated hydroxytoluene (BHT) were inactive. Similarly, morin, fisetin, kaempferol and n-propyl gallate markedly inhibited TPA-caused ODC induction. Esculetin, BHA, α-naphthol, 2, 3-DHNA and α-tocopherol inhibited it less potently, but significantly. (+) Catechin, (-) epicatechin and BHT failed to inhibit or only slightly inhibited TPA-caused ODC induction. TPA-caused DNA synthesis was not inhibited by morin, esculetin, (+)-catechin or α-tocopherol. The TPA-induced skin tumor promotion was markedly inhibited by morin and slightly suppressed by esculetin and α-tocopherol, but (+)-catechin was inactive. Thus, the inhibitory effects of flavonoids and antioxidants on the TPA-caused ODC induction and tumor promotion were roughly parallel with their activities of lipoxygenase inhibition. These results further support our hypothesis that a lipoxygenase product(s) is involved in the mechanism of TPA-caused ODC induction and tumor promotion.

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Variation of the binding Ulex europaeus agglutinin-I (Ulex-I), a lectin specific for blood group H (O) substance, was examined in normal, dysplastic and cancerous esophageal epithelium from 43 instances of carcinoma by means of the lectin-antilectin peroxidase-antiperoxidase method. The normal epithelium showed strong staining at the cell border, although the basal cells were entirely negative. In mild and moderate dysplasia, staining was negative, while in some cases of severe dysplasia positive staining was found in the basal cells. Most of the basal cells in carcinoma in situ revealed granular or diffuse positive staining in the cytoplasm. The results indicate that the loss of blood group H antigen occurs during carcinogenesis of the esophageal mucosa. Thus, Ulex-I may be a useful marker of cytoplasmic differentiation of the esophagus.

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Gastric cancer specimens obtained from 162 patients who had undergone radical surgery with the routine postoperative administration of both mitomycin C and 5-fluorouracil were stained for carcinoembryonic antigen (CEA) by the unlabeled antibody enzyme technique. The CEA (-/+) group (92 cases) consisting of the negative and weakly positive staining cases had a significantly better survival rate over a period of 5 years than the CEA (++) group (70 cases) which comprised only strongly positive cases. The CEA (-/+) group with differentiated adenocarcinoma had the best prognosis and the 5-year survival rate was significantly higher than those of the other three groups. Among stage II and III carcinomas, the postoperative survival rate was significantly better in the CEA (-/+) group than in the CEA (++) group. Among the patients with lymph node metastasis, the postoperative survival rate was low, especially in the CEA (++) group. The present data suggest that staining for CEA in tissue sections of stomach carcinoma may be helpful in differentiating among tumors that appear similar by conventional histological methods, thus providing a new means for obtaining more precise prognostic information.

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Antimetastatic activity of cell-wall skeleton of Propionibacterium acnes C7 (P. acnes-CWS) in C57BL/6 mice varied depending on the injection route. The kinetics of antimetastatic effect against Lewis lung carcinoma (3LL) revealed that the sooner intratumoral (it) injection of P. acnes-CWS was carried out, the better the result. However, intravenous (iv) injection of P. acnes-CWS produced the best result if P. acnes-CWS was injected at about the time when metastases began to develop in the lungs. Pretreatment with intrafootpad (ifp) injection of P. acnes-CWS inhibited primary tumor growth and subsequent pulmonary metastases especially when given 7 days before tumor inoculation into the same footpad, but tended to enhance artificial pulmonary metastases when given 7 days before iv injection of tumor cells. In contrast, pretreatment with iv injection of P. acnes-CWS enhanced spontaneous pulmonary metastases especially when given 7 days before ifp inoculation of tumor cells, but inhibited artificial pulmonary metastases when given one day or 7 days before iv injection of tumor cells. These results suggest the difficulty of treatment of tumor metastases with immunological adjuvants. In T-cell-deprived mice, it injection of P. acnes-CWS showed no antimetastatic effect against 3LL, but iv injection was still effective. This indicates that T-cells are required for the antimetastatic effect of it injection of P. acnes-CWS, but iv injection of P. acnes-CWS is able to inhibit pulmonary metastases in T-cell-deprived mice.

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Twenty-eight drugs, most of which are tertiary amines, were tested for the formation of volatile nitrosamines by reaction with nitrite under physiological conditions; the drugs (10mM) were incubated with nitrite (40mM) at pH 3.0, 37° for 1 and 4hr. The volatile nitrosamines formed were determined by gas chromatography-thermal energy analysis. Of the 28 drugs, 24 formed measurable amounts of volatile nitrosamines that are known carcinogens. The yields of nitrosodimethylamine (NDMA) from aminopyrine (55-65%) and minocycline (11%) were higher than that from dimethylamine under the same conditions. This result suggests that there may be a pathway not involving the secondary amine (dimethylamine) as an intermediate in the formation of NDMA from minocycline as well as from aminopyrine, Tolazamide gave rise to nitrosopiperidine (NPIP) in addition to nitrosohexamethyleneimine (NHXI), formation of which was expected from the chemical structure of tolazamide, and the yield of NPIP (2-7%) was higher than that of NHXI (0.2-1.2%). Ascorbic acid (40mM) was effective in decreasing the formation of nitrosamines from drugs by reaction with nitrite, although the blocking effects varied between 88 and 100% depending on the drugs tested or on the nitrosamines formed.

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DMG, a new polysaccharide with a well-characterized structure, isolated from the culture filtrate of an actinomycetes and then degraded by acid treatment, was tested for antitumor activity on allogeneic and syngeneic tumors in mice. In the allogeneic Ehrlich solid tumor system, DMG showed antitumor activity over a wide dose range, its optimal dose being 10-100mg/kg. The optimal time of DMG administration was 1-2 weeks after tumor inoculation, but DMG was also effective when given before tumor inoculation. DMG was effective when given ip, sc, it (intratumorally) or iv. DMG also had antitumor effects on syngeneic tumors. It rapidly inhibited the growth of MM46 mammary carcinoma, MH134 hepatoma, and Meth A fibrosarcoma, and also inhibited spontaneous pulmonary metastases of B16-BL6 melanoma. However, it had no direct cytocidal action on tumor cells in vitro. Its antitumor activity was much less in athymic nude mice and in mice immunosuppressed by whole-body X-irradiation than in normal hosts. Thus, DMG was shown to exert antitumor activity via host-mediated mechanisms. Its antitumor activity is discussed in comparison with those of other antitumor polysaccharides.

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The immunopharmacological behavior of DMG, an antitumor polysaccharide, was studied in mice. DMG administered ip or sc stimulated peritoneal macrophages to produce high levels of interleukin-1 activity, which can amplify successive immune responses. DMG dose-dependently and schedule-dependently increased the cellular immune response against allogeneic tumor cells and the humoral immune response to sheep erythrocytes. DMG also enhanced nonspecific antitumor effector functions, such as natural killer activity of spleen and peritoneal cells, and the cytostatic activity of peritoneal macrophages. Peritoneal macrophages activated by ip or sc injection of DMG exhibited high cytostatic activity, especially after exposure in vitro to lymphokine supernatants containing macrophage activation factor. Moreover, granulocyte/macrophage colony-stimulating activity in the serum increased 2-10hr after DMG administration. Thus, DMG potentiated antigen-specific immunological functions and nonspecific functions of host defense systems against cancer both qualitatively and quantitatively.

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A cytolytic factor partially purified from eggs of the sea hare Aplysia kurodai was examined for cytolytic activity against various target cells. All kinds of tumor cells tested were lysed in vitro by the cytolytic factor in the range of 10-100ng protein/ml. In contrast, normal spleen cells were lysed by 10μg/ml of this factor and red cells were not lysed even at this higher concentration. Tumor lysis was time-dependent and was complete within 10hr. This factor inhibited DNA and RNA syntheses of tumor cells but not protein synthesis. The cytolytic activity was lost on heat-treat-ment (60°) and at pH2, and was partially inhibited by treatment with 8M urea and at pH12, but the factor was resistant to treatments with trypsin, periodate and 2-mercaptoethanol. Neutralizing activity was observed in vivo on pretreatment of MM46 and L1210 cells with the factor. This cytolytic factor also inhibited the growth of solid-type MH134 tumor and ascitic-type MM46 tumor. These results indicate that Aplysia eggs contain a novel cytolytic factor that lyses tumor cells in vitro and inhibits tumor growth in vivo.

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Forty-two human hematopoietic cell lines were assessed for sensitivity to a fourday incubation with L-asparaginase. Eight of 13 T-leukemia cell lines and 1 of 7 non-T, non-B common acute lymphoblastic leukemia cell lines exhibited an ID₅₀ (50% growth inhibition dose) of less than 0.0001IU/ml. Seven of these sensitive cell lines were further cultured in L-asparagine-free medium and were found not to proliferate. Five T-leukemia cell lines, 6 non-T, non-B common acute lymphoblastic leukemia cell lines, 10 “abnormal” B-cell lines, 4 “normal” B-cell lines, 3 myeloma cell lines and 5 myeloid leukemia cell lines had ID₅₀ values between 0.1 and 1.0IU/ml. Twelve of these resistant cell lines were able to proliferate in L-asparagine-depleted medium The results indicate that some patients with T-cell acute lymphoblastic leukemia might respond to this enzymatic antineoplastic drug, L-asparaginase, at low dose.

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The role of platelets in cancer metastasis was studied by investigating the effects of the antiplatelet agents ticlopidine, diltiazem, dipyridamole and trapidil on artificial and spontaneous pulmonary metastases in mice. These agents were tested at their optimal inhibitory doses on adenosine diphosphate-induced platelet aggregation; namely, 100mg/kg for ticlopidine, 2mg/kg for diltiazem, 180mg/kg for trapidil and 60mg/kg for dipyridamole. At these doses, trapidil caused moderate inhibition of thrombin-induced platelet aggregation in mice, but the other agents had only slight effects. Artificial pulmonary metastasis was produced by inoculation of Lewis lung carcinoma (LLC) or B16 melanoma (B16) cells into C57BL/6 mice. For induction of spontaneous pulmonary metastases, these tumor cells were implanted subcutaneously into the footpads of mice. The resulting primary tumors of LLC and B16 were removed 9-10 and 17 days later, respectively. Artificial pulmonary metastases were inhibited significantly by all the antiplatelet agents tested. Spontaneous pulmonary metastases were markedly reduced only when these agents were given after removal of the primary tumor. The role of platelets is discussed with respect to thrombus formation in the lodgement of tumor cells and the participation of platelet-derived growth factor in the growth of metastatic foci.

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The pharmacokinetics of human recombinant interferon-β (ReIFN-β) was compared with that of natural human interferon-β (IFN-β) in monkeys and rabbits after intravenous or intramuscular injection. After intravenous injection of 10⁶units/kg of ReIFN-β or IFN-β into monkeys or rabbits, serum levels of both interferons declined biexponentially. No significant differences between ReIFN-β and IFN-β were detected in most pharmacokinetic parameters including T1/2-β, though T1/2-α of ReIFN-β was significantly shorter than that of IFN-β. These results seemed to be in conflict with the observed difference of stability of the interferons in vitro since ReIFN-β was less stable than IFN-β in monkey and rabbit serum. When ReIFN-β (10⁷ units/kg) was injected intramuscularly into monkeys or rabbits, it remained detect. able in the serum for 24hr; an absorption phase and an elimination phase were seen. However, AUC (the area under the serum concentration curve) after the intramuscular injection of ReIFN-β was about one-half and one-third of that after the intravenous injection in monkeys and rabbits, respectively. ReIFN-β and IFN-β (10⁶units/kg) were both detectable in the serum after intramuscular injection into rabbits, but the level of ReIFN-β was lower than that of IFN-β. These results indicate that the lack of carbohydrate in ReIFN-β did not essentially affect the in vivo pharmacokinetics in monkeys and rabbits after intravenous injection, but this was not the case after intramuscular injection.

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