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Title: Induction of mutagenesis and transformation in BALB/c-3T3 clone A31-1 cells by diverse chemical carcinogens

Abstract

BALB/c-3T3 cells were employed to examine the genotoxic potential of a variety of known chemical carcinogens. BALB/c-3T3 cells displayed a dose-dependent transformation response to a variety of carcinogens (polycyclic hydrocarbons, methylating agents, ethylating agents, aflatoxin B{sub 1} (AFT{sub 1}), and 4-nitroquinoline-N-oxide (4-NQO)). When the ability of these compounds to induce mutagenesis to resistance to the cardiac glycoside ouabain (OUA{sup R}) was examined, the authors found the short chain alkylating agents to be particularly effective mutagens, causing biologic effects at doses below those necessary to induce a transformation response. In contrast, the polycyclic hydrocarbons which were potent transforming agents were weaker, albeit significant, mutagens for the OUA{sup R} locus in this system, while AFB{sub 1} was quite weak. Further studies were performed with 5-azacytidine (5-AZA) and the nongenotoxic carcinogen cinnamyl anthranilate (CIN). 5-AZA was a potent transforming agent, but failed to cause mutagenesis. CIN similarly caused in vitro transformation. When a series of eight structurally diverse compounds were examined in both the BALB/c-3T3 and C3H10T1/2 mouse fibroblast transformation systems, the BALB/c-3T3 system was shown to be sensitive to a wide variety of potential carcinogens, whereas the C3H10T1/2 system proved routinely sensitive only to the polycyclic hydrocarbons.

Authors:
 [1]; ; ; ;  [2]
  1. (National Cancer Institute, Frederick, MD (USA))
  2. (Microbiological Associates, Bethesda, MD (USA))
Publication Date:
OSTI Identifier:
5840058
Resource Type:
Journal Article
Resource Relation:
Journal Name: Environmental and Molecular Mutagenesis; (USA); Journal Volume: 16:1
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; CARCINOGENS; GENETIC EFFECTS; FIBROBLASTS; ONCOGENIC TRANSFORMATIONS; AFLATOXINS; BENZOPYRENE; MUTAGENESIS; NITROSO COMPOUNDS; POLYCYCLIC AROMATIC HYDROCARBONS; POLYCYCLIC NITRO COMPOUNDS; QUINOLINES; ANIMAL CELLS; ANTIGENS; AROMATICS; AZAARENES; AZINES; BIOLOGICAL EFFECTS; CELL TRANSFORMATIONS; CONDENSED AROMATICS; CONNECTIVE TISSUE CELLS; HETEROCYCLIC COMPOUNDS; HYDROCARBONS; MATERIALS; NITRO COMPOUNDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PYRIDINES; SOMATIC CELLS; TOXIC MATERIALS; TOXINS; 560300* - Chemicals Metabolism & Toxicology

Citation Formats

Lubet, R.A., Kouri, R.E., Curren, R.A., Putman, D.L., and Schechtman, L.M. Induction of mutagenesis and transformation in BALB/c-3T3 clone A31-1 cells by diverse chemical carcinogens. United States: N. p., 1990. Web. doi:10.1002/em.2850160103.
Lubet, R.A., Kouri, R.E., Curren, R.A., Putman, D.L., & Schechtman, L.M. Induction of mutagenesis and transformation in BALB/c-3T3 clone A31-1 cells by diverse chemical carcinogens. United States. doi:10.1002/em.2850160103.
Lubet, R.A., Kouri, R.E., Curren, R.A., Putman, D.L., and Schechtman, L.M. 1990. "Induction of mutagenesis and transformation in BALB/c-3T3 clone A31-1 cells by diverse chemical carcinogens". United States. doi:10.1002/em.2850160103.
@article{osti_5840058,
title = {Induction of mutagenesis and transformation in BALB/c-3T3 clone A31-1 cells by diverse chemical carcinogens},
author = {Lubet, R.A. and Kouri, R.E. and Curren, R.A. and Putman, D.L. and Schechtman, L.M.},
abstractNote = {BALB/c-3T3 cells were employed to examine the genotoxic potential of a variety of known chemical carcinogens. BALB/c-3T3 cells displayed a dose-dependent transformation response to a variety of carcinogens (polycyclic hydrocarbons, methylating agents, ethylating agents, aflatoxin B{sub 1} (AFT{sub 1}), and 4-nitroquinoline-N-oxide (4-NQO)). When the ability of these compounds to induce mutagenesis to resistance to the cardiac glycoside ouabain (OUA{sup R}) was examined, the authors found the short chain alkylating agents to be particularly effective mutagens, causing biologic effects at doses below those necessary to induce a transformation response. In contrast, the polycyclic hydrocarbons which were potent transforming agents were weaker, albeit significant, mutagens for the OUA{sup R} locus in this system, while AFB{sub 1} was quite weak. Further studies were performed with 5-azacytidine (5-AZA) and the nongenotoxic carcinogen cinnamyl anthranilate (CIN). 5-AZA was a potent transforming agent, but failed to cause mutagenesis. CIN similarly caused in vitro transformation. When a series of eight structurally diverse compounds were examined in both the BALB/c-3T3 and C3H10T1/2 mouse fibroblast transformation systems, the BALB/c-3T3 system was shown to be sensitive to a wide variety of potential carcinogens, whereas the C3H10T1/2 system proved routinely sensitive only to the polycyclic hydrocarbons.},
doi = {10.1002/em.2850160103},
journal = {Environmental and Molecular Mutagenesis; (USA)},
number = ,
volume = 16:1,
place = {United States},
year = 1990,
month = 1
}
  • This study provides a preliminary comparative evaluation of the responses to a series of 49 chemicals, in in vitro transformation assays, of Balb/3T3 cells, Syrian hamster embryo cells, and Fischer 344 rat embryo cells infected with Rauscher murine leukemia virus. The chemicals assayed included aromatic amines; polycylic aromatic hydrocarbons; alkylating agents; nitrosamines, hydrazines, and related compounds; heterocyclic compounds, amides, ureas, and acylating agents; inorganic compounds; and hormones. In all three assays 37 of the chemicals were tested. The most uniform test responses were obtained with the polycyclic aromaic hydrocabons and inorganic compounds. With the other groups of chemicals, more variationmore » in response was observed. This study expands the base of information on the potential of these in vitro transformation systems, and the lack of responses with some of the chemicals underscores the need for incorporation of exogenous metabolic activating systems into these assay systems.« less
  • In the process of in vitro cell transformation, normal cells, which have an oriented pattern of growth and a limited life span in vitro and which are not tumorigenic, are converted into cells that have a hereditary random pattern of growth, the ability to grow continuously in culture, and the ability to form tumors. Such heritable phenotypic changes may arise from alterations in gene expression due to somatic mutations after interaction of the carcinogen with cellular DNA. Our studies have indeed shown (a) that metabolically activated carcinogenic polycyclic hydrocarbons which have been shown to bind to cellular DNA induce somaticmore » mutations in mammalian cells; (b) that there is a relationship between the degree of mutant induction and the degree of carcinogenicity of the different hydrocarbons tested; and (c) that the somatic mutations were induced by metabolites rather than by the hydrocarbons themselves. In the case of benzo(a)pyrene (BP), a very common carcinogenic polycyclic hydrocarbon, its 7,8-diol-9,10-oxide was identified as the major mutagenic and cell-transforming metabolite. Based on these studies, it was possible to estimate the genetic target size for cell transformation by comparing in the same cells the frequency of cell transformation and mutation for ouabian resistance (which is presumably due to a mutation at one locus) induced by BP and by one of its major metabolites. The results indicated that the target size for transformation is 20 times larger than that determined for ouabain resistance. This suggests that cell transformation, as determined by a hereditary pattern of cell growth, may be due to a mutation and that this mutation can occur in one out of a small number of the same or different genes.« less
  • The mouse cell line MO-5 is resistant to transformation by various chemical carcinogens and also by UV irradiation. Northern (RNA) blot analysis showed active expression of ras and myc genes in MO-5 and BALB/3T3 cells. The effect of transfection of various oncogenes on transformation was compared in MO-5 cells and parental BALB/3T3 cells. Activated c-H-ras, c-N-ras, and v-mos gene induced transformation foci of MO-5 and BALB/3T3. Introduction of the polyomavirus middle T-antigen (mTag) or the Rous sarcoma virus-related oncogene v-src, however, efficiently transformed BALB/3T3 but not MO-5 cells. Expression and phosphorylation of mTag and the associated c-src proteins were observedmore » in mTag-transfected clones of MO-5 as in BALB/3T3 and phosphorylation of the src protein was observed in v-src-transfected BALB/3T3 and MO-5 clones. Hybrids between mTag- or v-src-induced transformants of BALB/3T3 and untransformed MO-5 maintained the transformation phenotype, suggesting that no dominant suppressor of transformation exists in MO-5. A hybrid clone between BALB/3T3 and MO-5 induced efficient transformation foci after transfection with the mTag gene, suggesting that the deficient transformation phenotype of MO-5 was recessive. Instead, some other alteration of MO-5, plausibly membrane function, might lead to abortive transformation by chemical carcinogens and also by mTag and the v-src gene product.« less
  • The response of BALB/3T3 clone A31-1-1 cells to chemically induced morphological transformation was evaluated using 3-methylcholanthrene (MCA). Stock cultures were initiated from cryopreserved cells, grown in T25 flasks containing 5 ml of medium, and replated at subconfluency. Serially transferred cells were then subjected to transformation assay. After 24-hr seeding, cells were incubated 48 hr with MCA in a 5% CO/sub 2/ incubator. They were then rinsed and incubated for an additional 4 weeks with twice weekly medium change. Type III foci were scored after fixation and staining with Giemsa. With serial passage from the frozen state, cells of passages 3-14more » had a low level of spontaneous transformation; zero to 6 type III foci per 20 dishes were counted. In the MCA-treated cultures the number of transformed foci, however, increased with passage. Such passage-related sensitivity to MCA was demonstrated for cells cultured in two batches of sera: one from MA Bioproducts (Lot no. 2E052) and the other from Armour Pharmaceuticals (Lot no. Y65801). The passage-related increase in number of transformed foci was not related to doubling time, cloning efficiency, or MCA-induced growth inhibition.« less
  • We have developed a cell-mediated mutagenesis assay in which cells with the appropriate markers for mutagenesis are co-cultivated with either lethally irradiated rodent embryonic cells that can metabolize carcinogenic hydrocarbons or with primary rat liver cells that can metabolize chemicals carcinogenic to the liver. During co-cultivation, the reactive metabolites of the procarcinogen appear to be transmitted to the mutable cells and induce mutations in them. Assays of this type make it possible to demonstrate a relationship between carcinogenic potency of the chemicals and their ability to induce mutations in mammalian cells. In addition, by simultaneously comparing the frequencies of transformationmore » and mutation induced in normal diploid hamster cells by benzo(a)pyrene (BP) and one of its metabolites, it is possible to estimate the genetic target size for cell transformation in vitro.« less