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~' 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.

Members of another class of powerful direct mutagens, four nitroazabenzo[a]pyrene derivatives, were identified by Sera and co-workers (1994) in the basic fraction of extracts of diesel exhaust and in ambient POM. Structures and direct mutagenic activities on strains TA98 and YGf024 (-S9 mix) of the f- and 3-nitro-6-azabenzo[a]pyrenes and the f- and 3-nitro-6-azabenzo[a]pyrene-/V-oxides are shown in Table 10.2f (Sera et al., f992; Fukuhara et al., 1992). They have been measured in ambient air in Fukuoka, Japan, at concentrations of 1.1, 1.2, 0.8, and 0.3 ng/g, respec tively, and in diesel emissions at concentrations of 4.9, 7.7, 2.2, and 3.8 ng/g, respectively (Sera et al., 1994).

Another powerful direct mutagen identified in ambient particulate matter collected in Santiago, Chile, is 3,6-dinitrobenzo[a]pyrene. Although its concentration is low (0.002 ng m~3 of air), its specific direct activities on strains TA98 and YGf024 (-S9 mix) are high, 400,000 and 4,800,00 rev ¡jlg"1, respectively (Sera et al, 1991). For a discussion of polar forms of BaP in ambient particles, see Ismail et al. (1998).

g. Microbiology and Analytical Chemistry: Bioassay-Directed Fractionation and Chemical Analysis of Complex Mixtures of POM

Analysis of the complex mixtures of gaseous and/or particulate POM in primary emissions or ambient air is a daunting task, given the huge numbers of species and the small concentrations. The development of the Salmonella typhimurium assay has helped to direct such analysis through the technique of bioassay-directed chemical analysis.

With this approach, fractionation by a suitable HPLC technique separates the organic extract of a complex mixture of POM into subtractions of increasing polarities. These are then assayed for mutagenicity, e.g., using the microsuspension modification of the Ames assay. The resulting mutagram shows how the "total" mutagenic activity of the original POM extract is distributed among several chemically distinct classes of mutagenic subtractions. Conventional analytical techniques are then employed to determine the chemical structures and concentrations of the mutagenic species present in each subtraction.

Finally, one bioassays each of the newly identified agents and compares the sum of their mutagenic potencies with the total specific activity of the original complex POM mixture. These quantities may or may not be equal, depending on several factors, including the possibility of synergistic or antagonistic effects due to chemical interactions (e.g., see Donnelly et al., 1998). For examples of the use of bioassay-directed fractionations and chemical analysis, see Epler et al. (1979), Guerin et al. (1979), Huisingh et al. (1979), Pellizzari et al. (1979), Epler (1980), Pitts et al. (1980), Waters et al. (1979, 1981, 1983), Bj0rseth (1983), Holmberg and Ahlborg (1983), Alfheim et al. (1983, 1984a, 1984b), Thilly et al. (1983), Schuetzle et al. (1985) and references therein, Nishioka et al. (1985, 1988), Salmeen et al. (1984, 1985), Schuetzle and Lewtas (1986), Lewtas et al. (1990a, 1990b), Arey et al. (1992), Harger et al. (1992), Zinbo et al. (1992), Greenberg et al. (1993), Sasaki et al. (1995), and Gupta et al. (1996).

TABLE 10.21 Direct Mutagenicities of Two Nitroazabenzo[a]pyrenes, Their N-Oxide Derivatives," and 3,6-Dinitrobenzo[a]pyrenec Detected in Ambient and Diesel Particulate Matter'' and, for Comparison, 1- and 3-Nitrobenzo[a]pyrene



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