Plate incorporation assay

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. 

TABLE 6.7. Positive Controls for Use in Plate Incorporation Assays... 

Several statistical approaches have been applied to the results of plate incorporation assays (Wahrendorf et al., 1985 and Mahon et al., 1989). These authors make a number of important suggestions to maximize the power of statistical analyses those that relate to the method of analysis are reproduced below. 

The use of controls is as described for the plate incorporation assay. It is crucial to use the minimum amount of organic solvent in this assay, as the total volume of the incubation mixture is small relative to the solvent component. 

TABLE 10.16 Direct Mutagenicities of Selected Nitro-PAHs in the Salmonella typhimurium Plate Incorporation Assay ( — S9) with Strains TA98, TA98NR, and TA98/ l,8-DNP6u,b... 

A brief outline of the essential features of the standard plate incorporation assay (Maron and Ames, 1983) is given in Box 10.9. Protocols for pure chemicals and for complex mixtures of primary emissions and ambient aerosols are also described by Belser et al. (1981) and Alfheim et al. (1984b) intra- and interlaboratory comparisons are discussed later in this chapter. 

Specific activities of individual airborne PAHs and PACs determined by the standard plate incorporation assay cover a huge range e.g., the mono- and dinitro-PAHs in Table 10.16 have direct activities ( — S9) ranging from several hundred to 10A rev pg. This is also true for different isomers of the same compound. For example, the 1-, 3-, and 6-N02-BaP isomers formed in laboratory exposures of BaP particles to N02 (plus a trace of HN03) in air have direct activities (TA98, — S9) of 2500, 5300, and <1 rev /xg 1, respectively (Pitts et al., 1984b). 

Compound MW Microsuspension assay, TA98 — S9 (rev nmol 1) Plate incorporation assay, TA98 — S9 (rev nmol 1)... 

While present only in relatively small amounts in ambient aerosols (e.g., 5.2-11.5 pg m 3), its mutagenicity, 208,000 rev nmol-1 in TA98 (Salmonella typhimurium, plate incorporation assay with preincubation, - S9 mix) and 6,290,000 rev nmol-1 in strain YG1024, increases its importance in contributing to the total mutagenicity. 

Reddy, J.K., Moody, D.E., Azamoff, D.L. Rao, M.S. (1976) Di-(2-ethylhexyl)phthalate an industrial plasticizer induces hypolipidemia and enhances hepatic catalase and carnitine acetyltransferase activities in rat and mice. Life Sci., 18, 941-945 Reddy, J.K., Reddy, M.K., Usman, M L, Lalwani, N.D. Rao, M S. (1986) Comparison of hepatic peroxisome proliferative effect and its implication for hepatocarcinogenicity of phthalate esters, di(2-ethylhexyl) phthalate, and di(2-ethylhexyl) adipate with a hypolipidemic drag. Environ. Health Perspect., 65, 317-327 Rexroat, M.A. Probst, GS. (1985) Mutation tests with Salmonella using the plate-incorporation assay. Prog. Mutat. Res., 5, 201-212 Rhodes, C., Orton, T.C., Pratt, I.S., Batten, P.L., Bratt, H., Jackson, S.J. Elcombe, C.R. (1986) Comparative pharmacokinetics and subacute toxicity of di(2-ethylhexyl) phthalate (DEHP) in rats and marmosets extrapolation of effects in rodents to man. Environ. Health Perspect., 65, 299-307... 

Rexroat, M.A. Probst, GS. (1985) Mutation tests with Salmonella using the plate-incorporation assay (Chapter 14). In Ashby, J., de Serres, F.J., Draper, M., Ishidate, M., Jr, Margolin, B.H., Matter, B.E. Shelby, M.D., eds. Progress in Mutation Research, Volume 5, Evaluation of Short-Term Tests for Carcinogens. Report of the International Programme on Chemical Safety s Collaborative Study on in vitro assays, Amsterdam, Elsevier Science Pubhshers, pp. 201-212... 

Baker, R. S. U. Bonin, A. M. (1985) Tests with the Salmonella plate-incorporation assay. Prog. Mutat. Res., 5, 177-180... 

Liquid plate incorporation assay cells transfected with rat GST 5-5+ were positive at 42 pg/mL ° Positive with mouse liver S9, negative with rat liver S9... 

To determine the mutagenic potential of nonaqueous liquids as measured by the Ames SaZmoneZ/a/mammalian-enzyme assay, the following protocol is recommended for the sample preparation. In step 1, the desiccator assay is performed on the neat material. The desiccator assay allows the detection of volatile mutagens (such as chlorinated solvents) that are often missed in the plate incorporation and pre-in-cubation assays (16, 17). In addition, a suspension of the neat material (20 mg/mL) is prepared by ultrasonication (5 min at room temperature) in high-purity DMSO (18, 19) and tested in the normal plate incorporation assay as well as in a pre-incubation Ames assay (20). The pre-in-cubation assay allows the detection of certain mutagens, such as dimethylnitrosamine, that require additional time for activation by mammalian or bacterial enzymes. A positive response in any of these three assays indicates the presence of mutagenic components, and the evaluation process is completed. 

Salmonella Mutagenicity Test (Ames Test). The methods of bacterial culture, the verification of genetic markers, and the plate incorporation assay were essentially the same as described previously (14, 15). Petri dishes (90 mm) containing about 20 mL of 1.2 Noble agar in minimal Vogel Bonner Medium E supplied with excess biotine and... 

Mercuric chloride was not mutagenic in the Salmonella typhimurium plate incorporation assay (Wong 1988). These negative results are not unexpected because the Ames test is not suitable for the detection of heavy metal mutagens. Oberly et al. (1982) reported, however, that doses of mercuric chloride (4.4 and 5.9 g Hg/mL) approaching severely cytotoxic levels induced a weak mutagenic response in mouse lymphoma L5178Y cells but only in the presence of auxiliary metabolic activation. 

Prival MJ, Mitchell VD. 1981. Influence of microsomal and cytosolic fractions from rat, mouse, and hamster liver on the mutagenicity of dimethylnitrosamine in the Salmonella plate incorporation assay. Cancer Res 41 4361-4367. 

Goto S, Endo O, Matsushita H. 1992. Results of a comparative study on the salmonella pre-incubation and plate incorporation assays using test samples for the IPCS collaborative study. Mutat Res 276 93-100. 

Pharmakon Europe (1994e) Test article ETBE. Salmonella typhimurium/mammalian microsome plate incorporation assay (Ames test). Report No 76593 for Elf, 18 April 1994... 

An aliquot of the total extract from each sample was tested in severe short-term bioassays as reported previously. The slope of the dose response curve (rev/yg) in the Salmonella typhimurium TA98 plate incorporation assay without metabolic activation (-S9) was utilized in this study to compare the nitro-aromatic content and direct-acting mutagenicity of each sample extract. 

In the plate Incorporation assay, S. typhimurium TA1535 tested positive at concentrations of 21, 190 and 1670 pg/plate, but only without S9. In the preincubatlon assay, S. typhimurium 00 tested positive only at 62 pg/plate and only with S9. 

Table 9.3. Plate incorporation assay for the Ames mutagenicity test for X -poly(GGAP). ...

The majority of the studies outlined in Section 2 utilized the rapid and inexpensive plate incorporation assay with the Salmonella histidine-reversion system.All strains (missense and frameshift) should be screened with unknown materials. However, in practice, most determinations can be made with strains TAIOO and TA98 (containing the R plasmid pkMlOl). Crude materials or fractions for testing are usually dissolved in DMSO in the range of 5-10 mg of total solids/ml. Stock solutions are stored in the dark at 4°C. Fractions or subfractions are evaporated or lyophilized to dryness or to a concentrated tar before solubilization. In actual practice, many crude fractions are not completely soluble even in DMSO. The treatment can be carried out with the suspended material or the DMSO-soluble materials after suspension and centrifugation. 

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