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A W Hsie

Publications and source records attributed to A W Hsie.

At least 55 records · Page 3Linked to original sources
PubMed1984

Detection of deletion mutations in pSV2gpt-transformed cells.

We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient CHO cells that have been transformed by the plasmid vector pSV2gpt. One isolated transformant, designated AS52, carries a single copy of the Escherichia coli gpt gene stably integrated into the high-molecular-weight DNA and expresses the bacterial gene for the enzyme xanthine-guanine phosphoribosyltransferase. Mutants deficient in this enzyme can be induced in the AS52 cell line by a variety of mutagens, and spontaneous or induced mutants can be selected for resistance to 6-thioguanine (Tgr). Two Tgr clones derived from the AS52 line were analyzed by Southern blot hybridization and were found to contain deletions involving at least a portion of the gpt gene. Because of the small size and stability of the integrated pSV2gpt plasmid, and the well-defined selection protocol for mutant isolation, the AS52 line offers promise as a system suitable for the study of mutation at the molecular level in CHO cells.

Animals↗
PubMed1983

Utilization of Chinese hamster cells in vitro and in vivo in genetic toxicology: a multiphasic approach.

An approach often used to study genetic risk is comparative mutagenesis in different organisms. We have begun the development and validation of a multiphasic genetic toxicity system using the Chinese hamster and its somatic cells to measure mutational events in the same (or similar) gene, the same chromosome derived from the same animal (Fig. 1). This system will eliminate much of the uncertainty generated when different mutational events in such evolutionally divergent organisms as bacteria, insects, and mammals are compared. Using CHO cells we have defined an assay, CHO/HGPRT, to determine mutation at the hgprt locus. Coupled with S9 metabolic activation system, the CHO/HGPRT assay can quantify gene mutation and cytotoxicity induced by various classes of chemicals, physical agents, and the combination thereof. The quantitative nature of this assay permits elucidation of the structure-activity relationship for a given class of direct-acting agents. By incorporating the cytogenetics of CHO cells into this assay we can simultaneously measure induced chromosome aberrations and SCE. Using the stable CHO/human hybrid cell line AL-J1 measurement of chromosome deletion and/loss can be also performed. In order to further expand the usefulness of this genetic toxicity system to the molecular and whole animal levels we have begun development and validation of two additional systems. To study the molecular events which may result in mutation we are developing a CHOpSVgpt system. A Chinese hamster system with treatment in vivo is being developed to study mutation at the hgprt locus, chromosome aberration, and SCE in spleen cells in vitro. The use of this multiphasic genetic toxicity system at the cellular, molecular, and animal levels may soon provide reliable and rapid identification of suspected environmental mutagens.

Alkylating Agents↗
PubMed1983

Mutagenicity and cytotoxicity of dimethyl and monomethyl sulfates in the CHO/HGPRT system.

It has recently been shown that coal fly ash collected from coal-fired plants contains dimethyl sulfate (DMS) and monomethyl sulfate (MS) at concentrations as high as 830 ppm. Both these compounds were tested in the CHO/HGPRT system, and it was found that only DMS was cytotoxic and mutagenic to CHO cells. On a molar basis, DMS is twice a mutagenic as methyl methanesulfonate (MMS). Under our treatment conditions, maximum mutation induction and cytotoxicity were obtained after approximately 1 h. The Mutagenic potency of DMS was more stable in aqueous solutions at 4 degrees C than at the ambient temperature of 22 degrees C, but was least stable in DMSO solutions at 22 degrees C. Near-ultraviolet (near-UV) light caused an approximately twofold decrease in the mutagenic and cytotoxic effects of DMS. Although DMS produced by coal combustion could be rendered innocuous by environmental agents in a short span of time, this compound could still pose a health risk to workers closely involved in coal-combustion technology.

Animals↗
PubMed1983

Mutagenicity and cytotoxicity of five antitumor ellipticines in mammalian cells and their structure-activity relationships in Salmonella.

The mutagenicity and cytotoxicity of five antitumor compounds (ellipticines) were investigated in the Chinese hamster ovary cell hypoxanthine-guanine phosphoribosyltransferase assay and in six strains of Salmonella. All five compounds (ellipticine, 9-methoxyellipticine, 9-hydroxyellipticine, 9-aminoellipticine, and 2-methyl-9-hydroxyellipticinium) were cytotoxic and mutagenic in the Chinese hamster ovary cell hypoxanthine-guanine phosphoribosyltransferase assay in the presence or absence of Aroclor 1254-induced rat liver S9, and all except the last compound were mutagenic in Salmonella. Based on the reversion pattern obtained in various frame-shift and DNA repair-proficient (uvrB+) or -deficient (uvrB) strains of Salmonella in the presence or absence of S9, the first three compounds appear to cause frame-shift mutations by both intercalation and covalent bonding with DNA; thus, these are classified as reactive intercalators. However, 9-aminoellipticine intercalates only weakly and may instead exert its mutagenic activity primarily (or exclusively) by forming a covalent adduct with DNA. Compared to the published antitumor data obtained in mice, the results in Salmonella and Chinese hamster ovary cells suggest that the ability of ellipticine, 9-methoxyellipticine, and 9-hydroxyellipticine to intercalate with DNA, induce frame-shift mutations, and cause cell killing is associated with and may be the basis for their antitumor activity. The observation that the ellipticines are mutagenic in mammalian cells suggests that these antitumor agents may be carcinogenic.

Alkaloids↗
PubMed1982

Cytotoxicity and mutagenicity of dimethylnitrosamine in mammalian cells (CHO/HGPRT system); enhancement by calcium phosphate.

The cytotoxicity and mutagenicity of dimethylnitrosamine (DMN) was determined in the CHO/HGPRT system. Metabolic activation of the promutagen was achieved by use of liver homogenate supernatant (S9) prepared from Aroclor 1254-induced Sprague-Dawley rats. The cytotoxic and mutagenic effects of DMN were enhanced by the inclusion of calcium chloride in the incubation mix, and this enhancement was dependent on the presence of sodium phosphate. Under conditions that yielded maximal activity (10 mM calcium chloride, 10 mM magnesium chloride, 50 mM sodium phosphate), an apparent calcium phosphate precipitate was observed. DMN activity increased with increasing amounts of S9 protein over the range of 0.3-3.0 mg/ml in the S9 mix and appeared to plateau at higher concentrations. The mutagenicity of DMN can be described as 110 mutants/10(6) cells per mM DMN per mg/ml S9 protein per hour.

Animals↗
PubMed1982

Phenotypic expression time of mutagen-induced 6-thioguanine resistance in Chinese hamster ovary cells (CHO/HGPRT system): expression in division-arrested cell cultures.

The phenotypic expression time of ethyl methanesulfonate (EMS) induced 6-thioguanine-resistant mutants was studied with Chinese hamster ovary cells in culture (CHO/HGPRT system). After mutagen treatment of exponential phase cultures, the cells were maintained either in the exponential phase through subculture in medium containing 5% dialyzed fetal bovine serum (FBS) or in a nondividing viable state by use of medium containing 0-1% dialyzed FBS. The time course of expression of the 6-thioguanine-resistant phenotype was similar with both exponential phase and division-arrested cultures showing maximum expression by 9 days after mutagen treatment, and both methods of expression also yielded similar mutant frequencies over a range of EMS concentrations. This study shows that once the mutagenic event is fixed, the expression of the mutant phenotype does not require continued cell division since it occurs in division-arrested cultures. These results also suggest that both dilution of pre-existing hypoxanthine-guanine phosphoribosyl transferase (HGPRT) enzyme by cell division and turnover by protein degradation are involved in the phenotypic expression. Both processes occur in exponential cultures, but only protein turnover in arrested cultures. Consistent with this was the demonstration that the rates of total cell protein turnover increased in division-arrested cultures maintained in serum-free medium. These results separate genetic damage and phenotypic expression in a temporal sense, and point out the need to consider the mechanisms responsible for each process involved in the induction and expression of mutations.

Animals↗
PubMed1982

Quantification and analysis of reverse mutations at the hgprt locus in Chinese hamster ovary cells.

We describe an assay for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10(6) cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10 muM) is then added directly to the growing culture and AS-resistant (ASr) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT- clones. The ASr phenotype is characterized both physiologically and biochemically. All ASr clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT. The data provide further evidence that ICR-191 and ICR-170 cause structural gene mutations in mammalian cells and also suggest that ICR-191, ICR-170, and N-ethyl-N-nitrosourea induce similar types of mutations in Chinese hamster ovary cells.

Animals↗
PubMed1982

Effect of metabolic activation on the cytotoxicity and mutagenicity of 1,2-dibromoethane in the CHO/HGPRT system.

When ethylene dibromide (EtBr2) was assayed with the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system coupled with a rat liver metabolic activation system (S9), which contains Ca2+ (Ca, Mg-S9), the cytotoxicity of EtBr2 was greatly increased over that obtained when NADP was omitted from the Ca, Mg-S9 or when EtBr2 was assayed as a direct-acting agent. However, on a molar basis, the mutagenicity of EtBr2 remained unaffected. The omission of Ca2+ from the Ca, Mg-S9 metabolic activation system (Mg-S9), with either the addition or omission of NADP, caused approximately a 2-fold decrease in the mutagenicity of EtBr2 when compared to the results obtained by using the Ca, Mg-S9 system. The cytotoxicity of EtBr2 was further increased when a purified microsomal fraction, prepared from the S9 fraction, was used in the presence of Ca2+. In the absence of this calcium ion, this metabolic activation system was extremely cytotoxic to Chinese hamster ovary cells even without the presence of a mutagen or promutagen. The cytotoxicity of EtBr2 in the following assay systems decreased in this order: Ca, Mg-microsomes greater than Ca, Mg-S9 greater than S9 greater than direct-acting agent greater than or equal to Ca, Mg-S9 without NADP greater than or equal to Mg-S9 without NADP. Cytotoxicity appears to be NADP-dependent on the presence of NADP in the S9 system, the mutant yield (number of mutants that could be induced) was higher in its absence. Addition of reduced glutathione to Mg-S9 without NADP increased the mutagenicity of EtBr2 to values that did not exceed those obtained when EtBr2 was tested as a direct-acting agent. On a molar basis, ethyl methanesulfonate (EMS) is less cytotoxic but equally as mutagenic as EtBr2. However the mutant yield of EMS was higher than that of EtBr. Inclusion of Ca, Mg-S9 in the assay system had no effect on the biological activities of EMS.

Animals↗
PubMed1981

Cytotoxicity and mutagenicity of the fungicides captan and folpet in cultured mammalian cells CHO/HGPRT system).

The cytotoxicity and mutagenicity of the fungicides captan and folpet were determined in the CHO/HGPRT system which utilizes Chinese hamster ovary cells and resistance to 6-thioguanine to estimate mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus. Treatment of cultures with each compound for 5 hr is serum-free medium resulted in reproducible, significant, concentration-dependent increase in the frequency of 6-thioguanine-resistant mutants.

Animals↗
PubMed1981

Quantitative analysis of cytotoxicity and mutagenicity of benzo[a]pyrene in mammalian cells (CHO/HGPRT system).

Chinese hamster ovary (CHO) cells in cell culture were utilized to study the cytotoxic and mutagenic activity of benzo[a]pyrene (BP). A liver homogenate (S9) prepared from Arochlor 1254 induced male Sprague-Dawley rats was employed to mediate the biotransformation of the promutagen. The dependence of the induction of 6-thioguanine(TG)-resistant mutants on parameters such as BP concentration, amount of S9 protein, ionic composition of the S9 mix and treatment time was determined. Under optimal conditions with a 5-h treatment time, BP mutagenicity can be described as 185 mutants/10(6) cells per microM BP per mg/ml S9 protein.

Animals↗
PubMed1981

Effect of calcium phosphate and alumina C gamma gels on the mutagenicity and cytotoxicity of dimethylnitrosamine as studied in the CHO/HGPRT system.

When CaCl2 was added in increasing concentrations to a rat liver metabolic activation system (S9) buffered with sodium phosphate, the mutagenic activity and cytotoxicity of dimethylnitrosamine (DMN) in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system were greatly increased. This effect was not observed with an S9 mix buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). The calcium phosphate gel precipitate of the S9 mix possessed approximately 1/3 of the total activity of the mix, while the supernatant had only slight activity. However, when the calcium phosphate gel precipitate of a solution of S9 salts (without S9 protein) was added to the supernatant, the remaining 2/3 of the activity was recovered. Commercially obtained calcium phosphate, tricalcium phosphate, and alumina C gamma gels could substitute for CaCl2 in the S9 mix, but diethylaminoethyl cellulose (DEAE cellulose) could not. Alumina C gamma gel can exert its effect in the absence of both CaCl2 and phosphate in the S9 mix. Increasing the time of contact between the S9 protein and the S9 salts increased the efficacy with which the S9 mix activated DMN; this is indicative of an adsorptive process by calcium phosphate gel.

Alum Compounds↗
PubMed1981

Fluctuation analyses of spontaneous mutations to 6-thioguanine resistance in Chinese hamster ovary cells in culture.

Fluctuation analyses of the spontaneous appearance of 6-thioguanine (TG)-resistant mutants in cultured Chinese hamster ovary (CHO) cells were performed to investigate (1) whether the resistance is induced by the selective agent or is the result of a mutation which occurs prior to the TG selection and (2) to estimate the spontaneous mutation rate at the hypoxanthine--guanine phosphoribosyl transferase (hgprt) locus. The potential problem of phenotypic delay was minimized by allowing an adequate expression time through maintenance of the cultures in a division-arrested, viable state. The results demonstrate that the TG-resistant (TGr) cells arise randomly in the cultures, independently of the selective agent, which is consistent with spontaneous mutations. The average values for mutation rate +/- standard deviation, based on 4 independent determinations and 2 methods of calculation, are 3.4 +/- 1.2 X 10(-7) (median method) and 5.1 +/- 1.8 X 10(-7) (mean method) mutants/cell/generation.

Animals↗
PubMed1981

Mutagenicity and cytotoxicity of haloethanes as studied in the CHO/HGPRT system.

When haloethanes were being tested as direct-acting agents in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system, ethylene dibromide (EtBr2) exhibited more cytotoxic and mutagenic activity than ethylene dichloride (EtCl2), and the mixed halogenated congener ethylene bromochloride (EtBrCl) had an intermediate effect. On a molar basis, the relative mutagenic activity of EtBr2 : EtBrCl : EtCl2 was approximately 100 : 6 : 1. Cell survival was reduced to 50% by approx. 3, 6 and 50 mM of EtBr2, EtBrCl and EtCl2, respectively, and declined precipitously with increasing concentrations of the haloethanes. When these 3 haloethanes were assayed in the presence of S9, there was a 5-25-fold increase in cytotoxicity; however, only EtBrCl and EtCl2 also showed a concomitant increase in mutagenicity of 4-fold. The mutagenicity of EtBr2 remained unchanged when assayed in the presence of S9. When NADP was omitted from the S9 mix, which contains a NADPH-regenerating system, the increase in cytotoxicity and mutagenicity observed with the complete S9 mix was abolished. EtBr2 was shown to possess a molar equivalent mutagenic activity to ethyl methanesulfonate under the conditions of the assay. The cytotoxicity of EtBr2 increased as the time of treatment increased up to 24 h, while mutation induction appeared to peak at around 5 h.

Animals↗