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Add: 0.5 ml mammalian liver Metabolising system, (+ S9)

Medium on plate supplemented with just enough histidine and biotin to allow a few cell divisions

Spontaneous His ~ His + reversions

Colonies = Direct Mutagen(s) present in sample

No growth of colonies = no mutagenic activity

Colonies = promutagen (s) present in sample

FIGURE 10.14 Diagram of procedure for the standard Ames Salmonella typhimurium reversion assay for chemical mutagens (Maron and Ames, 1983).

pies of POM collected from primary emissions and polluted ambient air contain both direct (e.g., ni-troarenes and 1-nitropyrene) and activatable (e.g., BaP) bacterial mutagens.

Dose-response curves are generated from the two sets of data ( + S9 and — S9) by running two series of plates prepared from a range of serial dilutions (duplicate or triplicate plates for each dilution) of the original sample. The slope of the ideally linear portion of this curve is the specific activity of a pure chemical and is expressed in units of revertants per microgram (rev £tg~') or revertants per nanomole (rev nmoU1).

The mutagenic potency of a POM extract is generally expressed in units of revertants per microgram (rev pg~1) of the dried organic extract of the sample (sometimes, however, as rev mg~' of the original particulate matter). The concentration of mutagens in ambient air (or synthetic laboratory atmospheres)

is the mutagen density, generally given in units of revertants per cubic meter of ambient air (or chamber) sampled (rev m~3).

It should be noted that in the forward mutation Salmonella typhimurium TM677 assay (see later discussion), mutagenic potency is generally expressed in units of (mutagen fraction X f05)//j,g of "equivalent organic carbon (EOC)," and mutagen density (i.e., mutagen concentration) in units of (mutant fraction X 105)/m3 of air. Assays are carried out with (for promutagens), and without (for direct mutagens), "postmitochondrial supernatant," +PMS and — PMS, respectively. Since this is equivalent to + S9 or — S9 in the Ames test, we use the latter abbreviation for both assays (see Hannigan et al., 1996, and references therein). In this book, unless otherwise specified, the term "bacterial mutagenicity" refers to data obtained with the more commonly used Ames Salmonella typhimurium reversion assay.

YG1024 is ~30 times more sensitive than TA98 to f,8-dinitropyrene (2,f50,000 vs 72,200 rev/nmol; -S9). For example, Legzdins and co-workers (1994) used the enhanced response of strain YG102f to determine the mutagenicities of f4 PAHs, and several nitroarenes, S-atom PACs, and O-atom PACs in the ambient air of Hamilton, Ontario, Canada.

b. Assay Procedure

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. (f984b); 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.f 6 have direct activities ( — S9) ranging from several hundred to ~ 10ft rev /jlg. 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 HNO-,) in air have direct activities (TA98, -S9) of 2500," 5300, and <1 rev ^g"1, respectively (Pitts et al., 1984b).

c. The "Microsuspension Modification"

Given the very small amounts of material found in atmospheric samples, improvements in the Ames assay that use smaller amounts of sample and/or have improved sensitivity are clearly desirable. Yahagi et al. (1977) described one such approach. Subsequently, Kado and co-workers (f983) reported an experimentally simple, but highly effective, modification, which is now widely used. The term "microsuspension modification" comes from the use of small volumes, ~0.2 mL. Increased numbers (~109) of bacterial cells are added to the sample being tested, along with ( + S9) or without ( —S9) liver homogenate mix. After incubation for 90 min at 37°C, it is processed according to the standard Ames test protocol.

Use of the Kado microsuspension preincubation modification has become increasingly popular, in large part because it provides a major increase in sensitivity compared to the standard plate incorporation assay (Maron and Ames, 1983) and requires less sample. As seen in Tables 10.17 and 10.18, this is especially true for the volatile nitroarenes 2-nitrofluorene and 1- and 2-nitronaphthalene; less enhancement is achieved with the larger nitroarenes, e.g., 2-nitrofluoranthene (Arey, personal communication). The increased sensitivity is also illustrated by an intercomparison of the "modified" vs standard plate assay using the reference complex mixtures SRM 1649, "Air Particles" (Box 10.3), and SRM 1650, "Diesel Particles." The amount of material needed to detect and quantify mutagenicity was improved by a factor of f0-20 and the sensitivity was increased by a factor of ~30 (Claxton et al., 1992a, 1992b; see also Bagley and co-workers (1992) for re-

TABLE 10.17 Comparison of Specific Mutagenic Activities of Several Standard Nitroarenes Determined on Strain TA98 (— S9) Using the Salmonella typhimurium Assay with and without the Kado Microsuspension Modification"-"

Compound

Mutagenic activity (rev nmol 1)

Microsuspension assay

Plate assay

2-Nitrofluorene

4100"

93"

2-Nitrofluoranthene

3500"

960"

1-Nitronaphthalene

30"

0.05,'' 0.4''

2-Nitronaphthalene

680"

0.2/' 0.9''

" Adapted from Arey (1998a).

h Cited in reviews by Rosenkranz and Mermelstein (1985a, 1985b). ' Kado et al. (1983, 1986, 1991).

" Adapted from Arey (1998a).

h Cited in reviews by Rosenkranz and Mermelstein (1985a, 1985b). ' Kado et al. (1983, 1986, 1991).

suits of a comparison study conducted in their laboratory).

This modification has been applied in a variety of studies, including the mutagenic activity of "fine" ambient air particles as a function of particle size (Kado et

TABLE 10.18 Direct Mutagenic Activities, TA98 (- S9), in the Salmonella typhimurium Assay (Microsuspension Preincubation) of Standard Samples of 14 Methylnitronaphthalene Isomers" and Selected Nitroarenes''

Nitroarene"

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