Cytotoxicity and mutagenicity of dimethylnitrosamine in mammalian cells (CHO/HGPRT system): Enhancement by calcium phosphate

Abstract

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.

Use of aroclor 1254‐induced rat liver homogenate in the assaying of promutagens in chinese hamster ovary cells

“…Aroclor 1254-induced rat liver homogenate (S9) is by far the most widely used exogenous activation system for in vitro mutagenicity assays, including the Sulmonella typhimuriumlhistidine reversion assay [Ames et al, 19731, the mouse lymphoma/ thymidine kinese assay [Amacher and Turner, 19801, as well as the CHOiHGPRT assay [Machanoff et al, 1981;O'Neill et al, 1982; this report]. We have made observations here that are of practical importance to the use of S9 in the CHO/HGPRT assay.…”

Acute, Multiple-Dose Dermal and Genetic Toxicity of Nu-3: A Novel Antimicrobial Agent

“…Cultures were purged of preexisting mutants by growth for 2 days in DMEM/F-12 medium supplemented with 10% FBS, 5 × 10 −6  M thymidine, 1 × 10 −5  M hypoxanthine, and 3.2 × 10 −6  M aminopterin. The test was performed according to the procedure of O'Neill and Hsie [13] and O'Neill et al [14, 15]. The cells which were purged of preexisting mutants were treated for 4 h with the test and control materials and then allowed an expression time of 7–9 days; the cells were then replated.…”

Value and limitation ofin vitrobioassays to support the application of the threshold of toxicological concern to prioritise unidentified chemicals in food contact materials

“…7: LEC obtained in mammalian cells (MLA hprt); Lowest Effective Concentrations (LEC), expressed in mass/L from (Green et al 1976;Couch et al 1978;Clive et al 1979;Kaden et al 1979;Phillips et al 1980;Gupta and Singh 1982;O'Neill et al 1982;Connor et al 1983;Thompson et al 1983;Zamora et al 1983;Oberly et al 1984Oberly et al , 1993Wangenheim and Bolcsfoldi 1988;Eliopoulos et al 1995;Speit and Hartmann 1995;Hakura et al 2003;Valentin-Severin et al 2003;Kawaguchi et al 2010;Smith et al 2013).…”