Resistance to Pseudomonas toxin A: a sensitive marker to screen for mutagenic substances using V79 cells.
The goal of this study was the development of a mutagenicity assay in V79 cells using Pseudomonas toxin A (PTA) as a selective agent and to compare it with the V79/hypoxanthineguaninephosphoribosyl transferase (HGPT) assay. PTA inhibits protein synthesis by covalently modifying elongation factor 2 (EF-2). Mutations in the EF-2 gene, in genes responsible for the modification of EF-2, and also in genes for the transport of the toxin into the cells, lead to PTA resistance. This involvement of several genes might explain the relatively high spontaneous mean mutation frequency of (122.7 +/- 38.8) x 10(-6) in growing cells (2 micrograms PTA/ml) as compared with (3.97 +/- 4.68) x 10(-6) in the HGPRT test (10 micrograms thioguanine/ml), where only mutations in the HGPRT gene lead to resistance. Methyl methanesulphonate, methyl nitroso urea and UV light were directly mutagenic in the V79/PTA assay. Aflatoxin B1 was mutagenic in the presence of an S9 mix. Benzidine, procarbazine, 2-acetylaminofluorene (2-AAF) and 4-acetylaminofluorene (4-AAF) were positive in the V79/PTA test when rat hepatocytes were used for metabolic activation. Using cells treated together with those used for the PTA assay, benzidine, 2-AAF and 4-AAF were negative in the V79/HGPRT test, while procarbazine was found to be positive under these test conditions. Hexamethyl phosphoramide (HMPA) was tested in the presence of rat hepatocytes and was negative in both test systems. Phorbol-12-myristate-13-acetate was highly cytotoxic, but did not induce mutations at the PTA resistance loci. In conclusion, it was demonstrated that the V79/PTA assay is an easy and sensitive method to screen for gene mutations in mammalian cells.
Suter
W
W
Sandoz Pharma Ltd, Drug Safety Assessment, Basle, Switzerland.
Negro
L
L
Barrera
I
I
Schneider
B
B
D036002:ADP Ribose Transferases; D016604:Aflatoxin B1 / Q000633:toxicity; D000818:Animals; D001427:Bacterial Toxins; D015415:Biomarkers; D002460:Cell Line; D006224:Cricetinae; D004351:Drug Resistance; D005098:Exotoxins / Q000494:pharmacology*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000633:toxicity; D008770:Methylnitrosourea / Q000633:toxicity; D009152:Mutagenicity Tests / Q000379:methods*; D009154:Mutation; D013755:Tetradecanoylphorbol Acetate / Q000633:toxicity; D014466:Ultraviolet Rays; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D016604:Aflatoxin B1; D001427:Bacterial Toxins; D015415:Biomarkers; D005098:Exotoxins; D007041:Hypoxanthine Phosphoribosyltransferase; D008741:Methyl Methanesulfonate; D008770:Methylnitrosourea; D013755:Tetradecanoylphorbol Acetate; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A