Patricia A. Brimer

The dose-response relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells

The frequency of ethyl methanesulfonate (EMS)-induced mutations to 6-thioguanine resistance in a Chinese hamster ovary cells done K1-BH4 was studied at many EMS doses including the minimally lethal range (0–100 μg/ml) as well as the exponential killing portion (100–800 μg/ml) of the survival curve. The mutation frequency increases approximately in proportion with increasing EMS concentration at a fixed treatment time. The pooled data for the observed mutation frequency, f(X), as a function of EMS dose X, is adequately described by a linear function f(X)=10−6(8.73+3.45 X), where 0≤X≤800 μg/ml. One interpretation of the linear dose-response is that, as a result of EMS treatment, ethylation of cellular constituents occurs, which is directly responsible for the mutation. Biochemical analyses demonstrate that most of the randomly isolated 6-thioguanine-resistant variants possess a highly reduced or undetectable level of HGPRT activity suggesting that the EMS-induced mutations to 6-thioguanine resistance affect primarily, if not exclusively, the HGPRT locus.

Acrylamide binding to the DNA and protamine of spermiogenic stages in the mouse and its relationship to genetic damage

Mice received an intraperitoneal injection of 14C-labeled acrylamide (AA) at an exposure of 125 mg/kg to equal that used in genetic studies carried out by Shelby et al. (1986). Subsequently, spermatozoa were recovered from the reproductive tracts of the animals over a 3-week period and assayed for the amount of bound AA. A strong increase in the level of binding occurred in late-spermatid to early-spermatozoa stages; these same stages are also genetically most sensitive to the action of AA. At all time points, alkylation of DNA within the sperm accounted for a very small fraction (generally less than 0.5%) of the total sperm-head alkylation. However, alkylation of protamine, a protein unique to sperm cells, was found to be correlated with total sperm-head alkylation and accounted for essentially all of the AA binding. Two radioactive adducts were found in hydrolysed protamine samples, one of which co-eluted with a standard of S-carboxyethylcysteine. Protamine alkylation appears to be a significant cause of acrylamide-induced genetic damage in spermiogenic cells of the mouse.

The dose-response relationship for ultraviolet-light-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster ovary cells

Exposure of Chinese hamster ovary (CHO) cells clone K1BE4 to ultraviolet (UV) light at doses up to 86 ergs/mm2 did not significantly reduce cell survival, but UV doses of 86–648 ergs/mm2 produced an exponential cell killing. Observed mutation frequency to 6-thioguannine resistance induced by UV increases approximately in proportion to increasing doses up to 260 ergs/mm2 in a range of 5–648 ergs/mm2 examined. The pooled data of mutation frequency f(X) as a function of dose X from 0–260 ergs/mm2 is adequately described by f(X)=10−6 (13.6+2.04 X). That the UVinduced mutations to 6-thioguanine resistance affects the hypoxanthineguanine phosphoribosyltransferase (HGPRT) locus is supported by the observation that all randomly isolated drugresistant colonies contained highly reduced or undetectable HGPRT activity.

Quantitative analyses of radiation- and chemical-induced lethality and mutagenesis in Chinese hamster ovary cells.

The Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) mutational assay selects for mutant clones, of which greater than 98% are deficient in HGPRT enzyme activity. This system sensitively determines the mutagenicity of a variety of chemical and physical agents. With all chemical mutagens studied, mutation induction increases linearly with increasing mutagen concentration, without an apparent threshold effect. Direct-acting alkylating agents have been used to study the interrelationships among chemical structure, cellular lethality, and mutagenic activity. Physical agents such as uv and X irradiation and various light sources have also been studied. Among a total of 42 agents with documented carcinogenic or noncarcinogenic activity, the correlation with mutagenic activity in the CHO/HGPRT system is 95.2% (40/42).

Further evidence for the genetic origin of mutations in mammalian somatic cells: The effects of ploidy level and selection stringency on dose-dependent chemical mutagenesis to purine analogue resistance in Chinese hamster ovary cells

Abstract Whether resistance to purine analogues 8-azaguanine (AG) and 6-thioguanine (TG) in mammalian cells is due to gene mutation or to epigenetic changes was investigated by an ethyl methanesulfonate (EMS) dose-dependent induced “resistance” to these analogues in two near-diploid (2N) and one tetraploid (4N) Chinese hamster ovary (CHO) cells. EMS produced higher cell killing in 2N than in 4N cells. In the 2N cells, EMS-induced mutations to TG (1.7 μg/ml) resistance increased approximately as a linear function of the dose from 0–400 μg/ml. However, EMS was ineffective in inducing such mutation in the 4N cells. These observations are consistent with the notion that the induced TG resistance arose as a result of mutation at the gene or chromosome level. In each cell type, both the “observed” spontaneous and the EMS-induced frequency to purine analogue resistance decreased with increasing concentration of purine analogues. However, among the “resistant” clones a high proportion of those selected at 1.2 and 3.0 μg/ml of AG, a small portion selected at 7.5 μg/ml of AG, and virtually none at 1.7 and 6.0 μg/ml of TG are capable of growth in medium containing aminopterin (10 μM). This suggests that, under less stringent selective conditions, some resistant variants were being selected through mechanisms not yet defined.

Ethylene oxide inhalation at different exposure-rates affects binding levels in mouse germ cells and hemoglobin. Possible explanation for the effect

Male mice were exposed to [3H]EtO by inhalation at different exposure rates (300 parts per million (ppm) of EtO for 1 h: 150 ppm for 2 h: 75 ppm for 4 h). The total exposure was fixed at 300 ppm-h. The amount of EtO binding to developing spermatogenic stages, to sperm DNA, to testis DNA and to hemoglobin was then measured as a function of the EtO exposure rate. Generally, as the exposure rate increased there was an increase in the amount of EtO binding to the targets. For example, alkylation of sperm from the caudal epididymides 6 d posttreatment, of DNA from the vas sperm (averaged over 4 time points), of testis DNA (90 min posttreatment), and of hemoglobin (averaged over 4 time points), was 2.0 +/- 0.2 (SD), 1.8 +/- 0.4, 2.9 +/- 0.3, and 1.5 +/- 0.1 times greater, respectively, after an exposure to 300 ppm for 1 h than after an exposure to 75 ppm for 4 h. The testicular DNA from animals exposed to 300 ppm of [3H]EtO for 1 h was also analyzed for the presence of N7-hydroxyethylguanine (N7HEG) and O6-hydroxyethylguanine (O6HEG). The half-life (T1 2) of the N7HEG in the testis DNA was calculated to be 2.8 d. This lesion was removed relatively rapidly from the testis DNA and was probably excised by enzymatic repair. No formation of O6HEG was detected in any of the testis DNA samples analyzed. Additional experiments showed that the exposure rate effect was the result of less total EtO being taken in by the mice over long exposure times compared to that taken in during shorter exposure times at higher concentrations. This result argues against the idea that the exposure rate effect is the result of physiological/enzymological changes affecting transport or metabolism of the chemical within the animals under different exposure rate conditions.

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.

Mutagenicity of alkaline constituents of a coal-liquified crude oil in mammalian cells☆☆☆★

Employing the CHO/HGPRT system, we have shown that the acetone fraction of a crude coal--liquid crude oil is the major contributor to the mutagenicity of synthetic oil. The system appears to be useful for determination of mutagenicity of organic mixtures and for corroboration of results from other biological assays.

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.

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.