Tester Strains

Mutagenicity. The AJ-nitrosamines, in general, induce mutations in standard bacterial-tester strains (117). As with carcinogenicity, enzymatic activation, typically with Hver microsomal preparations, is required. Certain substituted A/-nitrosamine derivatives (12) induce mutations without microsomal activation (31,33,34). Because the a-acetoxy derivatives can hydroly2e to the corresponding a-hydroxy compounds, this is consistent with the hypothesis that enzymatic oxidation leads to the formation of such unstable a-hydroxy intermediates (13) (118). However, for simple /V-nitrosamines, no systematic relationship has been found between carcinogenicity and mutagenicity (117,119—123). 

Reversion Tests Background. There are several excellent references describing the background and use of bacteria for reversion tests (Brusick, 1987 Gatehouse et al., 1990). Three different protocols have been widely used plate incorporation assays, treat and plate tests, and fluctuation tests. These methods are described in detail in the following sections. Fundamental to the operation of these tests is the genetic compositions of the tester strains selected for use. 

Genetic Make up of Tester Strains. The most widely used strains are those developed by Bruce Ames and colleagues which are mutant derivatives of the organism Salmonella typhimurium. Each strain carries one of a number of mutations in the operon coding for histidine biosynthesis. In each case the mutation can be reverted either by base-change or by frameshift mutations. The genotype of the commonly used strains is shown in Table 6.6. 

E. coli Tester Strains. Ames and colleagues have made an impressive contribution to mutagenicity testing by the development of the Salmonella microsome test and, in... 

The following characteristics of the tester strains should be confirmed at monthly intervals or if the internal controls of a particular experiment fail to meet the required limits ... 

Repeat step (3), using 0.5 ml of 8 percent S9 mix per 2.0 ml aliquot of soft agar in addition to the test compound and tester strain. The S9 mix is kept on ice during the experiment. 

McCann, J., Spingam, N.-E., Kobori, J. and Ames, B.N. (1975b). Detection of carcinogens as mutagens Bacterial tester strains with R factor plasmids. Proc. Natl. Acad. Sci. USA 72 979-983. 

Wilcox, R, Naidoo, A., Wedd, D.J. and Gatehouse, D.G. (1990). Comparison of Salmonella Typhimurium TA102 with Escherichia coli WP2 tester strains. Mutagenesis 5 285-291. 

Nitroso-/V,/V-diniclhylanilinc has been reported to be bactericidal and mutagenic to Salmonella typhimurium TA 100 tester strains. In addition, this compound has proved to... 

Figure 1. The "spot test. Each petri plate contains, in a thin overlay of top agar, the tester strain TA98 and, in the cases of plates C and D, a liver microsomal activation system (S-9 Mix). Mutagens were applied to 6-mm filter-paper discs which were then placed in the center of each plate (A) spontaneous revertants (B) furyl-furamide (AF-2) (1 fig) (C) aflotoxin Bi(l fig) (D) 2-aminofluorene (10 fig). Mutagen-induced revertants appear as a ring of colonies around each disc (3).

Figure 2. Mutagenicity of 7-OH-2-AAF before and after purificatioti on HPLC. Each dose level was incorporated into top agar along with bacterial tester strain TA1538 and S-9 Mix. Histidine revertants were scored after 48 hours incubation (3,18j.

McMahon RE, Cline JC, Thompson CZ. 1979. Assay of 855 test chemicals in ten tester strains using a new modification of the Ames test for bacterial mutagens. Cancer Res 39 682-693. 

Aminopyridine was not mutagenic in a variety of Salmonella tester strains with or without metabolic activation. ... 

Nitromethane was not genotoxic in Salmonella typhimurium tester strains. ... 

VCD has been found to be mutagenic in a number of bacterial tester strains in the presence and absence of mammalian microsomal metabolic activation. It induced direct sister chromatid exchange and chromosomal aberrations in cultured Chinese hamster ovary cells. ... 

Biggart NW, Costa M. 1986. Assessment of the uptake and mutagenicity of nickel chloride in 5a/w(we//a tester strains. Mutat Res 175 209-215. 

This assay employs a number of different strains of Salmonella typhimurium mutants that are histidine auxotrophs. The first tester strains developed and tested on extracts of diesel and ambient POM in the mid to late 1970s included TA1535, which responds to base-pair substitution chemical mutagens (e.g., /f -propiolac-tone), and TA1537 and TA1538, which respond to frameshift mutagens, e.g., BaP and other PAHs. 

Matsumoto, Y., S. Sakai, T. Kato, T. Nakajima, and H. Satoh, Long-Term Trends of Particulate Mutagenic Activity in the Atmosphere of Sapporo. 1. Determination of Mutagenic Activity by the Conventional Tester Strains TA98 and TA100 during an 18-Year Period (1974-1992), Environ. Sci. Technol., 32, 2665 -2671 (1998). 

Watanabe, M., M. Ishidate, Jr., and T. Nohmi, Sensitive Method for the Detection of Mutagenic Nitroarenes and Aromatic Amines New Derivatives of Salmonella typhimurium Tester Strains Possessing Elevated O-Acetyltransferase Levels, Mutat. Res., 234, 337-348 (1990). 

Gee, R, Sommers, C.H., Melick, A.S., Gidrol, X.M., Todd, M.D., Burris, R.B., Nelson, M.E., Klemm, R.C. Zeiger, E. (1998) Comparison of responses of base-specific Salmonella tester strains with the traditional strains for identifying mutagens the results of avahdation study. Mutat. Res., 412, 115-130... 

Dobias, L. (1980). Human blood mutagenidty for Salmonella typhinuuium tester strains after oral application of entizol, Mutat. Res. 77,357. 

Marnett, L.J., Hurd, H.K., Hollstein, M.C., Levin, D.E., Esterbauer, H. Ames, B.N. (1985) Naturally occurring carbonyl compounds are mutagens in Salmonella tester strain TA104. Mutat. Res., 148, 25-34... 

Simula, T.P, Glancey, M.J. Wolf, C.R. (1993) Human glutathione S -transferase-expressing Salmonella typhimurium tester strains to study the activation/detoxification of mutagenic compounds studies with halogenated compounds, aromatic amines and aflatoxin Bp Carcinogenesis, 14, 1371-1376... 

Pavlov, YI. Khromov-Borisov, N.N. (1981) Metabolic activation of promutagens in the yeast-microsome test. 1. Selection and construction of tester strains and methods of checking them. Sov. Genet., 17, 915-924 (in Russian)... 

Levin, D.E., Hollstein, M., Christman, M.F., Schwiers. E.A. Ames, B.N. (1982) Anew Salmonella tester strain (TA102) with A.T base pairs at the site of mutation detects oxidative mutagens. Proc. natl Acad. Sci. USA, 79, 7445-7449... 

Hakura, A., Tsutsui, Y., Mochida, H., Sugihara, Y, Mikami. T. Sagami, F. (1996) Mutagenicity of dihydroxybenzenes and dihydroxynaphtlralenes for Ames Salmonella tester strains. Mutat. Res., 371, 293-299... 

---