The effect of chemical carcinogens on DNA bypass replication and the development of in vitro and in vivo models for chemical mutagenesis
This study with the testing of a hypothetical mechanism whereby mammalian cells are able to replicate their DNA past polycyclic aromatic hydrocarbon DNA adducts. The second objective of this thesis work was to develop both in vivo and in vitro models to study the induction of mutations in a target human gene by chemical carcinogens from two different classes, polycyclic aromatic hydrocarbons and nitrosamines. To approach the hypothetical mechanism of bypass replication in mammalian cells, synchronized Chinese hamster ovary cells were treated with benzo(a)pyrene, 7{beta}, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDEI). Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. It was determined using agarose gel electrophoresis that the ligation of Okazaki size replication intermediates was blocked in BPDE I-treated, synchronized CHO cells. To study mutagenesis of a specific sequence induced by chemical carcinogens, the human c-Ha-ras proto-oncogene was transfected into the mouse fibroblast cell line, NIH 3T3. Transfected NIH 3T3 cell lines (HHRN 1-4) were isolated that had a low copy number of the human c-Ha-ras proto-oncogene and a non-transformed phenotype. Treatment of the HHRN cell lines with the nitrosamine, N-methyl-nitroso-N{prime}-nitroguanidine (MNNG) resulted in transformed NIH 3T3 foci. In vitro MNNG treatment of the plasmid, z-6, and transfection into NIH 3T3 cells led to the isolation of transformed cell lines.
- Research Organization:
- Arizona Univ., Tucson, AZ (USA)
- OSTI ID:
- 6905215
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ADDUCTS
ANIMAL CELLS
ANIMALS
AROMATICS
BENZOPYRENE
CHO CELLS
CONDENSED AROMATICS
CONNECTIVE TISSUE CELLS
DNA ADDUCTS
DNA REPLICATION
ELECTROPHORESIS
FIBROBLASTS
GENES
HYDROCARBONS
IN VITRO
IN VIVO
MAMMALS
MICE
MUTAGENESIS
NITROSO COMPOUNDS
NUCLEIC ACID REPLICATION
ONCOGENES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHENOTYPE
RODENTS
SOMATIC CELLS
VERTEBRATES