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Gene mutation, quantitative mutagenesis, and mutagen screening in mammalian cells: study with the CHO/HGPRT system

Description: We have employed CHO cells to develop and define a set of stringent conditions for studying mutation induction to TG resistance. Several lines of evidence support the CHO/HGPRT system as a specific-locus mutational assay. The system permits quantification of mutation at the HGPRT locus induced by various physical and chemical mutagens. The quantitative nature of the system provides a basis for the study of structure-function relationships of various classes of chemical mutagens. The intra- and interlaboratory reproducibility of this system suggests its potential for screening environmental agents for mutagenic activity.
Date: January 1, 1980
Creator: Hsie, A.W.
Partner: UNT Libraries Government Documents Department

Quantitative and molecular analyses of mutation in a pSV2gpt transformed CHO cell line

Description: Following NDA-mediated gene transfer we have isolated a cell line useful for studying gene mutation at the molecular level. This line, AS52, derived from a hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficient Chinese hamster ovary (CHO) cell line, carries a single copy of the E. coli xanthine-guanine phosphoribosyl transferase (XGPRT) gene (gpt) and exhibits a spontaneous mutant frequency of 20 TG/sup r/ mutants/10/sup 6/ clonable cells. As with HGPRT/sup -/ mutants, XGPRT/sup -/ mutants can be selected in 6-thioguanine. AS52 (XGPRT/sup +/) and wild type CHO (HGPRT/sup +/) cell exhibit almost identical cytotoxic responses to various agents. We observed significant differences in mutation induction by UV light and ethyl methanesulfonate (EMS). Ratios of XGPRT/sup -/ to HGPRT/sup -/ mutants induced per unit dose (J/m/sup 2/ for UV light and ..mu..g/ml for EMS) are 1.4 and 0.70, respectively. Preliminary Southern blot hybridization analyses has been performed on 30 XGPRT/sup -/ AS52 mutants. A majority of spontaneous mutants have deletions ranging in size from 1 to 4 kilobases (9/19) to complete loss of gpt sequences (4/19); the remainder have no detectable (5/19) or only minor (1/19) alterations. 5/5 UV-induced and 5/6 EMS-induced mutants do not show a detectable change. Similar analyses are underway for mutations induced by x-irradiation and ICR 191 treatment.
Date: January 1, 1983
Creator: Stankowski, L.F. Jr.; Tindall, K.R. & Hsie, A.W.
Partner: UNT Libraries Government Documents Department

Quantitative mammalian cell genetic toxicology: study of the cytotoxicity and mutagenicity of 70 individual environmental agents related to energy technologies and 3 subfractions of a crude synthetic oil in the CHO/HGPRT system. [Hamsters]

Description: Conditions necessary for quantifying mutation-induction to 6-thioguanine resistance, which selects for >98% mutants deficient in the activity of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) in a near-diploid Chinese hamster ovary (CHO) cell line, referred to as CHO/HGPRT system, have been defined. Employing this mutation assay, we have determined the mutagenicity of diversified agents including 11 direct-acting alkylating agents, 16 nitrosamines, 10 heterocyclic nitrogen mustards, 15 metallic compounds, 5 quinolines, 5 aromatic amines, 27 polycyclic hydrocarbons, 13 miscellaneous chemicals, 7 ionizing and non-ionizing physical agents. The direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine is mutagenic while its noncarcinogenic analogue N-methyl-N'-nitro-N-nitroguanidine is not. Coupled with the rat liver S/sub 9/-activation system, procarcinogens such as nitrosopyrrolidine, benzo(a)pyrene, and 2-acetylaminofluorene are mutagenic while their analogues 2,5-dimethylnitrosopyrrolidine, pyrene and fluorene are not. The assay appears to be applicable for monitoring the genetic toxicity of crude organic mixtures in addition to diverse individual chemical and physical agents. The quantitative nature of the assay enables a study of EMS exposure dose: the mutagenic potential of EMS can be described as 310 x 10/sup -6/ mutants (cell mg ml/sup -1/ h)./sup -1/ It is also feasible to expand the CHO/HGPRT system for quantifying cytotoxicity and mutagenicity to determination of chromosomal aberrations and sister chromatid exchanges in cells treated under identical conditions which allows a simultaneous study of these four distinctive biological effects.
Date: January 1, 1978
Creator: Hsie, A. W.; Neill, J. P. & San Sebastian, J. R.
Partner: UNT Libraries Government Documents Department

CHO/HGPRT mutagenicity assay. III. Adaptation for mutagen screening

Description: This assay system has been employed for a variety of quantitative studies of mutation induction in mammalian cells (Hise et al., 1978). The development of interest in utilizing this system for mutagen screening has prompted us to evaluate those aspects of the system which necessitate the largest time and monetary investments, and to investigate the possibilities of applying insights gained from our basic research program to optimize use of the system for routine testing procedures. This paper describe our progress in the areas of phenotypic expression time and the density dependent recovery of mutant colonies in selective medium.
Date: January 1, 1979
Creator: O'Neill, J. P. & Hsie, A. W.
Partner: UNT Libraries Government Documents Department

CHO/HGPRT mutagenicity assay. II. Genetic basis of 6-thioguanine resistance

Description: An essential aspect of any attempt to study mutation induction in mammalian cells is the demonstration that genetic alterations are the bases for the altered phenotypes. We have defined reproducible conditions for the selection of 6-thioguanine-resistant (TG/sup r) variants of Chinese hamster ovary (CHO) cells. It is known that many such variants contain altered forms of the enzyme hypoxanthine (Hx)-guanine phosphoribosyl transferase (HGPRT). Our question was whether every variant colony which develops under our conditions has properties consistent with a genetic alteration at the HGPRT locus. We considered other possible mechanisms for the TG resistance such as: (a) colony formation by wild-type cells which have escaped the toxic effects of TG as a result of the death of cells and release of purine bases and metabolites into the medium and/or as the result of depletion of medium TG due to inherent decay and cellular metabolism leading to a loss of selection stringency; (b) phenotypic resistance due to some transient, adaptive response in cellular physiology, such as increased rates of purine biosynthesis; (c) stable resistance as a result of mutations at other loci, such as alterations in TG uptake or alterations which affect intracellular levels of purine nucleotides or PRPP. Our conclusion from these studies is that the TG/sup r/ phenotype which is quantified by use of the CHO/HGPRT system does represent genetic alterations, and that we are performing quantitative mutagenesis is determinations with this system. (PCS)
Date: January 1, 1979
Creator: O'; Neill, J P & Hsie, A W
Partner: UNT Libraries Government Documents Department

Quantitative mammalian cell mutagenesis and mutagen screening: study with CHO cells

Description: The CHO/HGPRT system has been developed and defined for quantifying mutation induced by various physical and chemical agents at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells. In all direct-acting chemical mutagens studied, mutation induction increases linearly as a function of the concentration, with no apparent threshold. Some chemicals induce mutation at non-cytotoxic concentrations. The mutagenicity of ethyl methanesulfonate has been quantified as a function of exposure concentration x treatment time. The sensitive and quantitative nature of the system enables studies of the structure-activity (mutagenicity) relationships of various classes of chemicals, including alkylating agents, heterocyclic nitrogen mustards, and platinum compounds. When rat liver S/sub 9/-mediated metabolic activation is present, procarcinogens such as benzo(a)pyrene, 2-acetylaminofluorene, and dimethylnitrosamine are mutagenic, whereas their noncarcinogenic structural analogues pyrene, fluorene, and dimethylamine are not. The system has been shown to be useful in determining the interactive effects between physical and chemical agents, and in screening for mutagenicity of fractionated organic mixtures and industrial chemicals in both liquid and gaseous state. For the system to be used successfully in routine screening, further studies should be directed toward the development of a metabolic activation system suitable for a broad spectrum of chemicals, a sensitive and reliable statistical method, and an experimental design to determine compounds with low mutagenicity. The system has been expanded for determination of mutagen-induced chromosome aberration, sister-chromatid exchange, and micronucleus formation in addition to gene mutation and cytotoxicity; it can also be used to study inhibition of DNA synthesis. (ERB)
Date: January 1, 1979
Creator: Hsie, A.W.; O'Neill, J.P.; San Sebastian, J.R. & Brimer, P.A.
Partner: UNT Libraries Government Documents Department