methylnitronitrosoguanidine and Granuloma

An in vivo-in vitro approach to studying neoplastic development in carcinogen-exposed rat fibroblasts was evaluated. In the model described, oncomodulin (Mr 12,000; pI 3.9), a tumor-associated and Ca2+-binding protein, was used as a specific marker of malignant transformation. A rapidly proliferating granulation tissue was exposed in vivo or in vitro to potent carcinogens like N-methyl-N'-nitro-N-nitrosoguanidine and procarbazine. As an endpoint of transformation anchorage independent (AI) colony formation in the soft agar assay was chosen. Exposure to various doses of N-methyl-N'-nitro-N-nitrosoguanidine in vivo or in vitro, or to procarbazine in vivo, led to induction of AI, transformed cells. Exposure of the cells to various doses of procarbazine in vitro produced neither formation of AI cells in the agar nor expression of oncomodulin in extracts of the exposed cell population. Almost all of the chemically induced AI cell lines tested have been found to be tumorigenic in athymic mice. In contrast, a very low rate (zero to two colonies per 10(6) cells tested) of spontaneous AI populations derived from untreated cells. None of these control AI colonies yielded tumors. In our transformation assay the appearance of neoplastic phenotypes seems very rapid, probably due to the increased cell division at the time of carcinogen-exposure. Expression of oncomodulin was found in extracts of transformed cells harvested from agar colonies, derived from carcinogen-exposed granulation tissue, but not from normal, untreated fibroblasts, as shown by two-dimensional polyacrylamide gel electrophoresis and high-performance liquid chromatographic analysis, as well as 45Ca2+-transblot electrophoresis. The presence of oncomodulin in extracts of transformed cells correlates well with the chemically induced colony formation in the soft agar assay. Oncomodulin might be a suitable neoplastic marker to study chemical carcinogenesis.

Topics: Animals; Biomarkers, Tumor; Calcium; Calcium-Binding Proteins; Cell Survival; Cell Transformation, Neoplastic; DNA Replication; Fibroblasts; Granuloma; Kinetics; Male; Methylnitronitrosoguanidine; Neoplasm Proteins; Rats; Rats, Inbred Strains

The mutagenic and DNA-damaging activities of oxolinic acid and pipemidic acid were evaluated in the rat granuloma pouch assay. After oral administration at high doses both the chemicals produce DNA alkali-labile sites in granuloma tissue cells, and with oxolinic acid an increase of liver and kidney DNA elution rate was also observed. In contrast, after direct injection into the granuloma tissue, DNA damage was absent. This indicates that DNA lesions are induced by a stable intermediate presumably formed in liver and converted to the ultimate DNA-damaging species in extrahepatic tissues. The simultaneous analysis of 6-thioguanine-resistant cells in the granuloma tissue revealed no statistically significant mutation induction either after local treatment or oral administration. No clear dose-response curve was obtained. However, after oral application in some of the animals an enhanced mutation frequency was detected, and the cloning efficiency of cells exposed to the drugs was reduced even after culturing them for 6 days. The most likely explanation is that functional multilocus mutations are induced which cannot be recovered efficiently at the HGPRT locus.

Topics: Animals; Clone Cells; DNA; DNA Damage; Female; Granuloma; Liver; Methylnitronitrosoguanidine; Mutagenicity Tests; Mutagens; Nicotinic Acids; Oxolinic Acid; Pipemidic Acid; Rats; Rats, Inbred Strains

Topics: Animals; Antigens, Helminth; Granuloma; Larva; Lymphocytes; Methylnitronitrosoguanidine; Mice; Mice, Inbred C57BL; Ovum; Schistosoma japonicum

The mutagenic activity of the natural plant product aristolochic acid (AA) was tested in the Granuloma Pouch Assay, which detects gene mutations induced in a subcutaneous granuloma tissue of rats. After direct exposure of the target tissue, AA induced high frequencies of mutants at a relatively low cytostatic/cytotoxic level. AA was more potent that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at equimolar doses. After oral application of AA, a dose-dependent mutagenic activity was seen. In contrast a very weak and inconsistent mutagenic effect was seen after systemic application of MNNG. These observations suggest that after oral application AA is not detoxified efficiently and can exert its mutagenic activity in extrahepatic tissues whereas MNNG is detoxified to a large extent at the site of administration.

Topics: Administration, Oral; Animals; Aristolochic Acids; Drug Resistance; Granuloma; Hypoxanthine Phosphoribosyltransferase; Inactivation, Metabolic; Male; Methylnitronitrosoguanidine; Mutagenicity Tests; Mutagens; Phenanthrenes; Rats; Rats, Inbred Strains; Thioguanine

Proliferation of granulation tissue on the inside of a subcutaneous (s.c.) air pocket was induced in rats by administration of 0.5 ml of 0.25% croton oil (granuloma pouch). Two days after induction of the granuloma, i.e., during the period of maximal cell growth, a single dose of 0.6 mg or 0.1 mg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was administered into the pouch. Fibrosarcomas of various histopathological types developed in 87% of the rats receiving the high dose and in 64% of the low dose animals. The mean latency period was 47.5 and 51.7 weeks respectively. Only one local sarcoma developed in rats treated with 0.6 mg MNNG by the s.c. route, and no tumors were observed in the groups treated with 0.1 mg s.c. The appearance of local sarcomas in the granuloma pouch tissue is correlated with previously reported frequency of point mutations (OUAR and HGPRT-) induced in granuloma fibroblasts with the same doses of MNNG. Possible mechanisms explaining the marked enhancement of the carcinogenic effect of MNNG in the granuloma pouch assay are discussed.

Topics: Animals; Croton Oil; DNA; Dose-Response Relationship, Drug; Fibrosarcoma; Granuloma; Male; Methylnitronitrosoguanidine; Mutation; Rats; Sarcoma, Experimental