3-Methylcholanthrene triggers the differentiation of alveolar tumor cells from canine bronchial basal cells and an altered p53 gene promotes their clonal expansion.
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Alveolar type II cells arising in canine bronchial autografts following exposure to 3-methylcholanthrene (MCA) give rise to carcinomas of varying glandular and squamous growth patterns. To study the role of the tumor suppressor gene p53 in this process, sections from progressive lesions were immunostained for p53 protein; microdissected regions were screened for p53 mutations. Adjacent sections were examined for type II cell expression [cuboid cell shape, large roundish nucleus, cytoplasmic staining for surfactant protein A (SP-A)] and proliferating cell nuclear antigen expression. Evidence for an altered p53 function (nuclear staining, missense mutations) was found in most carcinomas of all histologic types and in all grades of bronchial dysplasia, but not in hyperplastic or normal bronchial epithelium. It was primarily associated with the hyperplastic type II cell populations present in the basal zone of the lesions. In addition, we found SP-A staining in hyperplastic (but not in normal) bronchial basal cells. These data suggest that MCA initiates type II cell differentiation through phenotypic selection (basal cells). Inactivation of the p53 gene promotes the clonal expansion of the type II cells into discernible populations of (squamous or glandular) alveolar tumor cells. This in vivo study is the first to show that p53 is involved in a specific pathway leading to bronchogenic carcinoma. (+info)
Spontaneous development of cytotoxic activity in cultured lymphnode cells from tumour-bearing rats.
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Incubation in vitro of lymphnode cells (LNC) from rats bearing a transplanted syngeneic methylcholanthrene-induced sarcoma (Mc7) resulted in the generation of a potent cytotoxic activity. Four to seven days' culture was required for development of cytotoxic activity, which was shown to be mediated by a heat-stable soluble factor. The cytotoxicity was not detectable in a 3 h or 15 h 51Cr-release assay, but was demonstrated in a 48 h microcytotoxicity assay, where post-labeling with isotopically labelled cell precursors was used to quantitate cell survival. The cytotoxicity of the cultured tumour-bearer LNC and their supernatant factor was shown to be cross-reactive for tumour cell lines other than sarcoma Mc7, and was also expressed against adult or embryonic fibroblasts. (+info)
Ozonation of mutagenic and carcinogenic polyaromatic amines and polyaromatic hydrocarbons in water.
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The Salmonella-microsome assay for mutagenesis was used to determine the effect of ozone on the mutagenesis of selected carcinogens and mutagens in water. Short periods of ozonation were shown to completely inactivate the mutagenicity of several polyaromatic amine mutagens including acriflavine, proflavine, and beta-naphthylamine. Selected polyaromatic hydrocarbons were also sensitive to ozonation. Kinetic studies revealed that the mutagenicity of benzo(a)pyrene, 3-methylcholanthrene, and 7,12-dimethylbenz(a)anthracene was destroyed after short periods of ozonation. To correlate loss of mutagenicity with loss of carcinogenicity, two polyaromatic hydrocarbons were treated with ozone, extracted from water with hexane, and tested for carcinogenicity in mice. When 7,12-dimethyl-benz(a)anthracene and 3-methyl-cholanthrene were treated with ozone, there was a substantial reduction in carcinogenicity compared to control groups treated with oxygen alone. However, a small number of tumors developed in the group of animals receiving a hexane extract of ozonated 7,12-dimethylbenz(a)anthracene. This activity may be due to breakdown products of 7,12-dimethylbenz(a)anthracene that are not mutagenic. (+info)
CD8+ T cells are crucial for the ability of congenic normal mice to reject highly immunogenic sarcomas induced in nude mice with 3-methylcholanthrene.
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An attempt was made to identify the selection pressures put upon a growing tumour by CD8+ T cells. To this end tumours induced with 3-methylcholanthrene in T cell-deficient nude mice and in congenic T cell-competent nu/+ mice were transplanted to nu/+ recipients. The rejection rate of the sarcomas from nude mice was almost twice that of the sarcomas from nu/+ mice. Depletion of CD8+ T cells from nu/+ recipients prior to transplantation made them accept nude tumours that were consistently rejected by untreated nu/+ recipients. These findings suggest that a methylcholanthrene sarcoma during its growth in a T cell-competent host adapts to the T cell system through a selective elimination of highly immunogenic tumour cells that are susceptible to CD8+ T cell-mediated lysis. (+info)
Factors affecting metabolism and mutagenicity of dimethylnitrosamine and diethylnitrosamine.
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For exploration of the factors affecting dimethylnitrosamine (DMN) mutagenicity, for gathering of information on the metabolism of DMN, a frequently used and relatively well-understood carcinogen, and for explanation of metabolic variations in DMN carcinogenicity, parallel in vitro assays of the microsomal activation of DMN to a mutagen and of DMN demethylation were performed. Salmonella typhimurium G46 reversions to histidine independence increase linearly with time of incubation for 30 min. At low concentrations of microsomal protein, increases in protein yield a more than proportional increase in mutations. Increasing DMN concentration saturates the enzyme, yielding less demethylation and fewer mutations proportionately. Mutagenesis is completely inhibited by 1 mM 2-diethyl-aminoethyl-2,2-diphenylvalerate. When both DMN and microsomal protein are varied at high concentrations, there is a simple linear relationship between mutagenicity and DMN demethylase activity. Thus DMN demethylase activity may be the primary controlling factor in the metabolism of DMN to a mutagen, and probably to a carcinogen; other simultaneous pathways of DMN metabolism proportional to demethylation have not been ruled out. Induction with both phenobarbital and 3-methylcholanthrene (3-MC) increased rat and mouse liver DMN demethylase activity. Mouse liver microsomes from the C57BL/6 strain demethylate DMN at a markedly lower rate than do microsomes from the C3H strain, but after 3-MC induction the relationship is reversed. Strain differences in activation of DMN were not found in the activation of diethylnitrosamine to a mutagen. Hepatic dealkylation of DMN and diethylnitrosamine to active mutagenic metabolites is increased in both rats and mice by both 3-MC and phenobarbital induction, which is in contrast to the findings of others that 3-MC and phenobarbital induction, which is in contrast to the findings of others that 3-MC decreases the incidence of DMN-induced hepatic tumors in rats, and phenobarbital decreases the incidence of diethylnitrosamine-induced hepatic tumors in mice. (+info)
Metabolism of cigarette smoke condensates by human and rat homogenates to form mutagens detectable by Salmonella typhimurium TA1538.
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Nineteen fractions of whole condensate of smoke from the University of Kentucky Reference Cigarette IRI were tested for mutagenicity in vitro using a bacterial indicator system. As little as 25 mug of the active fractions were mutagenic toward histidine-requiring Salmonella typhimurium TA1538, if the condensates were incubated in the presence of rat or human liver homogenates of lung were relatively inactive. Homogenates from livers of rats that had been treated with 3-methylcholanthrene converted condensates to mutagens more efficiently than did liver homogenates from man or from normal or phenobarbital-treated rats. Use of homogenates from animals treated with 3-methylcholanthrene gave much more reproducible results in smoke fraction assays because larger numbers of revertants were obtained, and dose-response curves were linear over the range 25 to 250 mug condensate. The linear dose-response curves permitted quantitative comparison of the various fractions. The mutagenicity per mg of basic fractions of whole smoke condensate is very high and that of neutral polycyclic hydrocarbons is very low. Because of the exquisite preferential sensitivity of the TA1538 test system to polycyclic amines and insensitively to alkyl polycyclics, there is a poor quantitative correlation between mutagenicity and carcinogenicity, as measured by skin painting or in vitro cell transformation. There is substantial evidence that many carcinogens are mutagens but that most of these compounds require metabolism before they are biologically active. If further development improves the sensitivity of the bacterial testing system to mutagenic derivatives of alkyl polycyclic and heteropolycyclic hydrocarbons, it may provide a convenient, rapid, quantitative, and inexpensive bioassay for the detection of potentially carcinogenic substances in tobacco smoke condensates. (+info)
Morphological transformation of C3H 10T1/2 cells by 99mTc-cardiolite.
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The induction of in vitro morphological transformation in C3H 10T1/2 cells by 99mTc-Cardiolite (contents of Cardiolite kit [hexakis(2-methoxyisobutylisonitrile) and other components] plus (99m)Tc generator eluate) was examined. METHODS: Cells were grown for 48 h in the presence of 99mTc-Cardiolite or decayed 99mTc-Cardiolite (99mTc-Cardiolite after 1 wk of storage), and cell survival and transformation were assessed by the colony-forming and focus assays, respectively. X-ray was used as a reference for radiation effects, and 20-methylcholanthrene was used as a positive control for focus formation. RESULTS: Exposure of cells to 99mTc-Cardiolite results in a transformation frequency that is not significantly different from that induced by the volume equivalent of decayed 99mTc-Cardiolite. The number of foci per viable cell increases linearly from approximately 0.17 x 10(-4) in the untreated control to 1.7 x 10(-4) at 37 kBq/mL and 30 x 10(-4) at 1100 kBq/mL 99mTc-Cardiolite or its decayed 99mTc-Cardiolite volume equivalent. Furthermore, exposure of cells to low extracellular concentrations of 99mTc-Cardiolite or decayed 99mTc-Cardiolite (cell survival, > or =88%) induces an approximately 20-fold greater number of transformants per viable cell than that observed after 0.5 Gy x-irradiation, a dose that causes the same level of toxicity. CONCLUSION: Radioactive and decayed 99mTc-Cardiolite induce morphological transformation of C3H 10T1/2 cells in vitro. The underlying mechanism does not seem to be related to the radiation effects of decaying 99mTc but to chemical(s) present in the 99mTc-Cardiolite kit. (+info)
Aromatic hydrocarbon receptor (AhR).AhR nuclear translocator- and p53-mediated induction of the murine multidrug resistance mdr1 gene by 3-methylcholanthrene and benzo(a)pyrene in hepatoma cells.
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The mouse multidrug resistance gene family consists of three genes (mdr1, mdr2, and mdr3) encoding P-glycoprotein. We show that the expression of mdr1 is increased at the transcriptional level upon treatment of the hepatoma cell line Hepa-1c1c7 with the polycyclic aromatic hydrocarbon 3-methylcholanthrene (3-MC). This increase is not observed in the aromatic hydrocarbon receptor (AhR)-defective TAOc1BP(r)c1 and the AhR nuclear translocator (Arnt)-defective BP(r)c1 variants, demonstrating that the induction of mdr1 by 3-MC requires AhR.Arnt. We show that the mdr1 promoter (-1165 to +84) is able to activate the expression of a reporter gene in response to 3-MC in Hepa-1c1c7 but not in BP(r)c1 cells. Deletion analysis indicated that the region from -245 to -141 contains cis-acting sequences mediating the induction, including a potential p53 binding sequence. 3-MC treatment of the cells increased the levels of p53 and induced p53 binding to the mdr1 promoter in an AhR.Arnt-dependent manner. Mutations in the p53 binding site abrogated induction of mdr1 by 3-MC, indicating that p53 binding to the mdr1 promoter is essential for the induction. Benzo(a)pyrene, a polycyclic aromatic hydrocarbon and AhR ligand, which, like 3-MC, is oxidized by metabolizing enzymes regulated by AhR.Arnt, also activated p53 and induced mdr1 transcription. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, an AhR ligand resistant to metabolic breakdown, had no effect. These results indicate that the transcriptional induction of mdr1 by 3-MC and benzo(a)pyrene is directly mediated by p53 but that the metabolic activation of these compounds into reactive species is necessary to trigger p53 activation. The ability of the anticancer drug and potent genotoxic agent daunorubicin to induce mdr1 independently of AhR.Arnt further supports the proposition that mdr1 is transcriptionally up-regulated by p53 in response to DNA damage. (+info)