Analytical methods in bioassay-directed investigations of mutagenicity of air particulate material

Mutation Research 636 (2007) 4–35 www.elsevier.com/locate/reviewsmr Community address: www.elsevier.com/locate/mutres

Review

Analytical methods in bioassay-directed investigations of mutagenicity of air particulate material Christopher H. Marvin *, L. Mark Hewitt National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario L7R 4A6, Canada Received 26 January 2006; received in revised form 1 May 2006; accepted 8 May 2006 Available online 18 March 2007

Abstract The combination of short-term bioassays and analytical chemical techniques has been successfully used in the identification of a variety of mutagenic compounds in complex mixtures. Much of the early work in the field of bioassay-directed fractionation resulted from the development of a short-term bacterial assay employing Salmonella typhimurium; this assay is commonly known as the Ames assay. Ideally, analytical methods for assessment of mutagenicity of any environmental matrix should exhibit characteristics including high capacity, good selectivity, good analytical resolution, non-destructiveness, and reproducibility. A variety of extraction solvents have been employed in investigations of mutagenicity of air particulate; sequential combination of dichloromethane followed by methanol is most popular. Soxhlet extraction has been the most common extraction method, followed by sonication. Attempts at initial fractionation using different extraction solvents have met with limited success and highlight the need for fractionation schemes applicable to moderately polar and polar mutagenic compounds. Fractionation methods reported in the literature are reviewed according to three general schemas: (i) acid/base/neutral partitioning followed by fractionation using open-column chromatography and/or HPLC; (ii) fractionation based on normal-phase (NP) HPLC using a cyanopropyl or chemically similar stationary phase; and (iii) fractionation by open-column chromatography followed by NP-HPLC. The HPLC methods may be preparative, semi-preparative, or analytical scale. Variations based on acid/base/neutral partitioning followed by a chromatographic separation have also been employed. Other lesser-used approaches involve fractionation based on ion-exchange and thin-layer chromatographies. Although some of the methodologies used in contemporary studies of mutagenicity of air particulate do not represent significant advances in technology over the past 30 years, their simplicity, low cost, effectiveness, and robustness combine to result in their continued application in modern laboratories. # 2007 Elsevier B.V. All rights reserved. Keywords: Air particulate; Bioassay-directed fractionation; Complex mixtures; Mutagenicity; Chemical characterization; Short-term bioassays; Salmonella; Genotoxicity; Fractionation

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Extraction methodology . . . . . . . . . . . . . . . . . 2.1. Extraction solvent. . . . . . . . . . . . . . . . . 2.2. Extraction method . . . . . . . . . . . . . . . . 2.3. Fractionation based on solvent extraction

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* Corresponding author. Tel.: +1 905 319 6919; fax: +1 905 336 6430. E-mail address: [email protected] (C.H. Marvin). 1383-5742/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2006.05.001

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Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Acid/base/neutral partitioning followed by fractionation . . . . . . 3.2. Normal-phase HPLC fractionation . . . . . . . . . . . . . . . . . . . . . 3.3. Open-column chromatography followed by normal-phase HPLC 3.4. Ion-exchange chromatography . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Thin-layer chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Mutation chromatograms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Other fractionation methodologies . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Many of the analytical methods used in environmental mutagenesis research are derived from techniques developed for the identification of specific, pre-determined, target analytes. However, the approach used in the determination of potential mutagens in complex environmental mixtures is fundamentally different. It is essential for effective strategies to combat the health effects of environmental mutagens to precisely characterize the nature of the compound, a mixture of compounds, or environmental factors that enhance or reduce mutagenicity. The combination of short-term

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bioassays with analytical chemical techniques has been successfully used in the identification of a variety of mutagenic compounds in complex mixtures. This marriage of chemical and biological tests is often referred to as ‘‘bioassay-directed chemical analysis’’ or ‘‘bioassay-directed fractionation’’ [1,2]. An example of a bioassay-directed fractionation scheme, applied in this instance for the investigation of mutagenic activity in an urban air particulate SRM (NIST SRM 1649), is shown in Fig. 1. In conventional environmental analytical chemistry, those compounds falling outside target compound classes are usually eliminated from the analyses by

Fig. 1. Bioassay-directed fractionation scheme applied to the investigation of mutagenic activity associated with an urban air particulate standard reference material (NIST SRM 1649) (reproduced from B.E. McCarry and D.W. Bryant, unpublished data). Discussion of mutagenic profiles of subfractions is provided in the text related to Fig. 2.

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fractionation and clean-up methods, including opencolumn chromatographic and high-performance liquid chromatographic techniques. In contrast, a full assessment of the mutagenic potential of an environmental sample requires that all of the material extracted from the matrix be tested for biological activity. Using bioassay-directed fractionation approaches, the crude extracts can be fractionated, and the resulting subfractions assayed to isolate mutagenic activity in mixtures of lesser complexity. Several iterative fractionation steps are usually required before mutagenic activity can be isolated in a manageable number of subfractions whose complexity is reduced to the point that structures for the candidate chemicals present can be postulated. Complete chemical confirmation of the proposed structures is obtained with authentic standards, which may require custom synthesis. Verification of mutagenicity is necessary to confirm the biological activity of the compounds identified, their relative potencies, their relative contributions to the overall potency of the matrix being examined, and any interaction effects that may be occurring. Subsequent to the confirmation of a causal mutagenic agent, more detailed studies for risk assessments can be conducted regarding such factors as environmental persistence, exposure, and metabolism. Analytical methods for bioassay-directed fractionation of any environmental matrix should ideally possess the following characteristics: 1. A high capacity in order to process the large quantities of extracted material required for full chemical and biological characterization. 2. Good selectivity for different compound classes. 3. The ability to ultimately resolve the large numbers of individual compounds within compound classes. 4. Be non-destructive in order to conserve the chemical and biological integrity of the analytes. 5. Compatibility with the scale and type of bioassay being used. 6. Reproducibility. It has been estimated that as much as 55–95% of the inter-laboratory variability in measurements of mutagenicity of air particulate samples is due to differences in analytical methods, rather than actual differences in mutagenicity of the samples [3–6]. Much of the early work in the field of bioassaydirected fractionation resulted from the development of a short-term bacterial assay employing Salmonella typhimurium; commonly known as the Ames assay. The Ames tester strains can be used to define the characteristics of the compounds causing the response

through their ability to induce different types of mutations (e.g., frame shift mutations vs. base-pair substitution mutations), and if there is a requirement for any metabolic activation to manifest a response (i.e., direct-acting mutagens vs. indirect-acting mutagens). The short-term nature and technically straightforward procedure of the Ames assay proved to be a natural complement to analytical methods, and resulted in a powerful tool for identification of environmental mutagens. During the 1970s and 1980s, new Salmonella strains were developed that provided more accurate characterization of mutagens. For example, strains were developed that were deficient in nitroreductase [7] or enhanced in nitroreductase (e.g., YG1021 and YG1026 developed by Watanabe et al. [8–10]) that resulted in reduced or increased responses to nitrated-PAHs, respectively. The availability of tester strains exhibiting sensitivities to individual compound classes has allowed the application of the Ames assay in a diagnostic context [11]. An example of the varying responses of specific compounds/compound classes to different Salmonella strains is shown in Fig. 2, which shows a comparison of the mutagenic profiles of NP-HPLC fractions prepared from air particulate from Hamilton, ON, Canada, and Washington, DC, USA, using the methodology shown in Fig. 1. This comparison is based on the conventional TA98 strain and TA98 transformed using plasmid pYG122, which confers high Oacetyltransferase activity with a resulting high sensitivity to aromatic amines and nitroarenes [10]. In the case of SRM 1649, the relative responses in the two strains are similar. However, for air particulate from Hamilton with its corresponding increased burden of nitroarenes due to industrial emissions, the mutagenic activity is greatly enhanced in the O-acetyltransferase strain in the nitroarene-containing fraction (N2). Bioassay-directed fractionation studies conducted in the late-1970s using the Ames assay focused on the determination of mutagens in diesel particulates [1], petroleum and petroleum substitutes, drinking water, and commercial products [12]. The protocol developed and reported by Schuetzle and Lewtas [1] in their overview of the application of bioassay-directed fractionation to environmental research, and an early review by Claxton [2] of fractionation and bioassay characterization methods used for air particulate, are useful for providing examples of the basic principles of the methodology. The primary steps in a bioassaydirected fractionation protocol usually include extraction, preparative fractionation, subfractionation, and chemical analysis at any of the stages in the fractionation procedure. Bioassays are also performed

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Fig. 2. Comparison of mutagenic profiles of NP-HPLC fractions prepared from air particulate from Hamilton, Ontario, Canada (left), and Washington, DC (right) using the methodology shown in Fig. 1 and bioassayed in TA98 (S9) and TA1538/DNP (S9) (reproduced from B.E. McCarry and D.W. Bryant, unpublished data).

at each step in the protocol in order to identify mutagenic fractions, and to ultimately isolate mutagenic compounds/compound classes, and to monitor overall recovery of mutagenic activity. These steps also reflect a primary advantage and disadvantage of the bioassay-directed fractionation method; this technique has the potential for identification of a wide range of genotoxicants; however, bioassays of multiple fractions can require prohibitively large quantities of extracted organic material [2]. Sequential extraction using solvents of varying polarities is frequently employed to maximize recovery of a wide range of organic materials. Examples of sequential extraction include the use of dichloromethane followed by methanol for extraction of mutagens from combustion emission samples, and dichloromethane followed by diethyl ether for extraction of mutagens from water samples [1]. Preparative and/or semi-preparative methods are often employed to maximize the yield of mutagenic material with which to conduct further fractionation and chemical analyses. Short-term bioassays such as the Ames microsome assay can require considerably greater masses of material (i.e., microgram or milligram quantities), compared to modern analytical chemical techniques that can detect analytes at the nanogram or picogram levels, or below. In addition, a positive response in the Ames assay requires validation through establishment of a dose–response relationship, which substantially increases the amounts of material required. Preparative or semi-preparative methods can include open-column,

high-performance liquid chromatography (HPLC), or partitioning into acidic, basic, and neutral fractions. Fractionation for the purpose of isolating mutagenic subfractions for detailed biological and/or chemical testing is usually accomplished using normal-phase (NP) HPLC and/or reversed-phase (RP) HPLC. The NP-HPLC stationary phase is polar in nature (e.g., silica) where solvents of increasing polarity represent increasingly ‘‘strong’’ elution potential. For example, hexane, dichloromethane, acetonitrile, methanol, and water represent increasingly stronger mobile phases in NP-HPLC. Conversely, RP-HPLC uses a nonpolar stationary phase (e.g., C18) where solvents of decreasing polarity represent increasingly stronger elution power. Correspondingly, each technique affords fractions containing analytes according to relative polarity. These HPLC methods offer rigorous, effective, and reproducible separations of extracts from a wide range of complex environmental mixtures. Preparative (  mg) scale separations are frequently conducted as a first step of fractionation, followed by semi-preparative or analytical scale (  mg) separations on active fractions. As with the preparative and the semi-preparative separations, the recovery of mutagenic activity (based on the potency of the crude extract) is monitored at each stage of the fraction procedure. The procedural recovery of both absolute mass of material and mutagenic activity can be achieved by summation of the masses and bioassay responses of the individual fractions/ subfractions, or through recombination of the fractions/ subfractions to afford a reconstituted parent fraction

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that is then weighed and assayed. Significantly high- or low mutagenic activities can result from synergistic or antagonistic effects. The fractionation procedure may also result in elimination of mutagenic compounds (low recovery of mutagenic activity compared to parent fraction), or removal of compounds toxic to the bioassay reagents (high recovery of mutagenic activity compared to parent fraction). In addition, chemical changes to compounds during any extraction or fractionation procedure may result in artificially high or low recovery of mutagenicity. Subsequent to preparative fractionation, the next step in the overall protocol that results in a more comprehensive fractionation into general chemical compound classes is sometimes referred to as ‘‘Level 1’’ fractionation. This level of fractionation may still be conducted on a semi-preparative scale, or may involve the use of analytical-scale HPLC columns, and can be accomplished using RP-HPLC or NP-HPLC. NP-HPLC has proven particularly effective for fractionation of polycyclic aromatic compounds (PAC) according to polarity. Work reported in the mid-1980s by Schuetzle and Lewtas [1] used NP-HPLC to fractionate extracts of diesel particulate material. PACs including 1-nitronaphthalene and 1,6-pyrene quinone were used as marker compounds to mark the elution boundaries between nonpolar, moderately polar, and polar fractions. These authors also found that classification of HPLC subfractions based on polarity allowed comparison of results from different laboratories using slightly different fractionation procedures [13]. In the same vein, Wise et al. [14,15] and May and coworkers [16,17] developed a widely used NP-HPLC method that affords separation of PAC of a wide range of polarities in addition to separating PAHs according to the number of aromatic rings. Variations of this method, e.g. [18], effectively separate relatively polar PAC such as ketoPAC and aza-PAC from their homocyclic PAH counterparts. This methodology is also capable of separating the PAH into isomeric molecular weight classes, e.g., according to the number of aromatic rings. Application of NP-HPLC in the Level 2 fractionation of air particulate samples including diesel particulate and urban air particulate results in satisfactory recovery of both mass and mutagenicity. However, although bioassay data obtained from subfractions may provide valuable information as to the nature of compounds responsible for observed biological activity, subsequent chemical analysis has also shown that these subfractions can still be extremely complex and be comprised of several hundred or several thousand compounds. Schuetzle et al. [19] identified a large number of

PAC in Level 1 subfractions of a diesel particulate standard reference material, including PAH substituted by hydroxyl, aldehyde, anydride, nitro, ketone, dinitro, and quinine functional groups. Despite the complexity of the Level 1 subfractions, some early bioassaydirected fractionation studies were successful in identifying potent mutagens, including 1-nitropyrene in diesel particulate [19], and dinitropyrenes (1,6- and 1,8-) in carbon blacks [12,20]. In the majority of cases, Level 1 subfractions are still far too complex to enable positive identification of individual mutagens. Subsequent subfractionation, sometimes referred to as ‘‘Level 2’’ fractionation, enables finer resolution of the components of the parent fraction. Analytical-scale RP-HPLC is frequently employed as a complement to the Level 1 NP-HPLC fractionation, resulting in an overall multi-dimensional chromatographic separation. The process of isolating individual compounds or compound classes is iterative, resulting in multiple levels of fractionation beyond Level 2. However, additional separation steps result in large numbers of subfractions for mutagenicity and chemical testing. Therefore, there are significant laboratory resource implications as a result of greater levels of fractionation. The level of fractionation that can be practically achieved is driven by the amount of sample initially extracted, and therefore the yield of extracted material. In addition, there are other constraints imparted by proceeding to Level 2 and beyond, including decreasing quantities of material for the bioassays, and additional difficulty in achieving satisfactory recoveries of mass and mutagenicity. Despite the additional cost in terms of time and resources, the combination of this type of fractionation with a short-term bioassay constitutes a very powerful tool for ultimately isolating and identifying environmental mutagens. In this respect, the bioassay essentially becomes another form of detector for the chromatographic separation. Superposition of the bioassay response on conventional forms of HPLC detection, such as ultraviolet absorption or fluorescence, affords what is called a ‘‘bioassay chromatogram’’ or ‘‘mutation chromatogram’’. Mutation chromatograms produced from Level 1 or Level 2 fractionations have in some cases resulted in isolation and subsequent identification of individual mutagenic compounds. Using this technique, Schuetzle and Lewtas [1] identified a series of seven mono- and dinitro-PAHs in mutagenic NP-HPLC subfractions (Level 1 fractionation) of extracts of light-duty diesel particulate; these compounds were responsible for 30– 40% of the total extract mutagenicity. Similarly, Marvin

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et al. [21] identified individual PAHs of molecular weights ranging from 252 to 302 amu in RP-HPLC subfractions (Level 2 fractionation) prepared using semi-preparative NP-HPLC (Level 1 fractionation) of coal tar-contaminated sediment; these PAHs accounted for the vast majority of the total mutagenicity exhibited by the crude extract. Compounds isolated in individual RP-HPLC subfractions exhibiting significant mutagenicity included benzo[a]pyrene, indeno[1,2,3-cd]pyrene, and benzo[ghi]perylene. Because of the implications for direct impacts of air quality on human health, air particulate material is one of the most widely studied matrices using bioassay-directed fractionation. Air particulate samples are also very complex due to the high number of chemical constituents, compared to most other environmental matrices. As a result, isolation of individual mutagens in subfractions, even after extensive fractionation, is a daunting task. For the purposes of this methods review, the term air particulate will encompass a number of different matrices including ambient air particulate, diesel particulate, airborne soots, and steel foundry air particulate. As part of this series, Claxton et al. [22] recently provided an excellent review of the mutagenicity of ambient outdoor air, including a section on methods for identification of mutagens. Earlier reviews covering the topics of extraction, chemical characterization, and bioassays of airborne particulate material include those of Hughes et al. [23], Chrisp and Fisher [24], and Epler [25]. A body of literature warranting special mention is volume 11 of Environment International published in 1985 [26]. The papers in this volume were derived from the Conference and Workshop on Genotoxic Air Pollutants convened in Rougemont, NC, in April 1984, the focus of which was the characterization of genotoxins in ambient air. Several of the papers published in this volume are cited in this review. A review by Dipple [27] of PACs as chemical carcinogens covers the earliest investigations of the identity of carcinogenic compounds in soots, tars, and pitches; these studies were precursors to bioassaydirected fractionation. The review by Chrisp and Fisher [24] covers the period up to 1980. The recent review by Claxton et al. [22] also contains a summary of the results of studies of airborne particulate, many of which are relevant to development of analytical methods, including 1. mutagenicity of airborne particulate is due to large numbers of compounds in varying compound classes; 2. preparation of complex environmental mixtures for bioassay must consider the questions being asked and compatibility with the bioassay;

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3. fractions of extracts containing PAHs exhibit increased mutagenic responses in the presence of metabolic activation (S9), while mutagenicity of fractions containing more polar compounds decreases with addition of S9; 4. there can be significant disparity between biological activities of extracts of airborne particulate and levels of PACs; 5. PAHs may not be the predominate class of mutagens present; 6. the majority of mutagenicity can be associated with moderately polar to highly polar compounds; 7. more polar fractions can contain nitroaromatics, aromatic amines, and aromatic ketones; 8. relative contribution of individual fractions to total mutagenicity can be influenced by the analytical fractionation scheme. In this paper, we have reviewed the subject of analytical methods used in bioassay-directed investigations of air particulate material with emphasis on mutagenesis as the endpoint. While other papers in this and previous volumes may have focused on comparative analyses of bioassay results for individual matrices, this paper serves to discuss issues related to determination of mutagens in air particulate material, including a critical review of the methods used for extraction and fractionation. This review does not cover analytical methods for determination of concentrations of genotoxins in air that are measured independently, and then correlated with mutagenic activity. While endpoints other than mutagenesis have investigated in bioassay-directed fractionation studies, the analytical approaches toward determining cause and effect for different endpoints are often identical. An excellent example of the isolation and identification of a causal agent was the investigation into the source of an outbreak of food poisoning in Canada in 1987 [28]. This investigation, which was concluded in less than 5 days, resulted in the identification of domoic acid in shellfish as the cause of the outbreak. This investigation, based on a bioassay-directed strategy generally similar to the studies outlined above, highlighted the importance of a number of factors, including extraction efficiency and use of a dose–response relationship in the bioassay. Despite significant differences in the physical, chemical and biological properties of environmental matrices including surface and drinking waters, effluents, air particulates, and soils and sediments, bioassay-directed investigations are based on common strategies. However, each of these matrices may present unique challenges and pitfalls that must be considered in

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developing methodology that allows the utility of shortterm bioassays, such as the Ames Salmonella microsome assay, to be fully exploited. 2. Extraction methodology 2.1. Extraction solvent Ultimately, the choice of optimum solvent system and/or technique for extraction is predicated on a number of factors, including matrix. Different types of air particulate samples can also have significantly different physical and chemical properties. The majority of methods developed for studies of air particulate material have used organic solvents to extract mutagens from the underlying solid matrix. The most common single extraction solvent has been dichloromethane [4,6,19,29–48,48–75]; other methods have employed methanol [56,76–79], acetone [80–94], hexane [95–97], benzene [98–101], cyclohexane [98,102–104], toluene [90], benzene/ ethanol [105–108], and benzene/methanol [109,110]. The works of Stanley et al. [111] and Gordon [112] represent some of the earlier studies of solvent efficacy in extraction of organic material from airborne particulate. Although these studies were not formally directed toward extraction of mutagenic activity, their basic findings are relevant to mutagenicity-based studies of air particulate. For example, Stanley et al. [111] in their review of extraction solvents for airborne particulate reported that use of acetone resulted in high extraction efficiency, but also afforded very complex extracts. Extraction with benzene yielded less-complex extracts, while cyclohexane resulted in less-complex, but PAH-rich, extracts. Similarly, Virgis et al. [104] used cyclohexane for extraction of airborne particulate sampled in Thessalonika, Greece, in order to minimize extraction of polar compounds. Stanley et al. [111] also reviewed the use of mixed-solvent systems, and concluded that advantages and disadvantages of individual solvents ultimately determine solvent selection. Gordon [112] related extraction efficiency to solvent characteristics including dielectric constant, solvent polarity, and solvent strength. Similar to Stanley et al. [111], Gordon found that moderate-strength solvents such as benzene resulted in good recovery of PAHs, while strong solvents (defined as having high e8 values) extracted greater quantities of polar organics; however, these solvents also extracted significant quantities of inorganic compounds. Møller and Lo¨froth [113] discouraged nonpolar solvents for extraction of mutagens from ambient air samples, and recommended use of dichloromethane or acetone. Singlesolvent systems using dichloromethane, methanol, and acetone were typically used for studies where investigators required a simple and robust system capable of extracting a wide range of compounds from airborne particulate. Dichloromethane was preferable for exclusion of non-organic components toxic to the bacteria used in bioassays. Other solvents were used to extract specific classes of compounds. For example, De Flora et al. [103] used cyclohexane to extract

PAHs from air particles. Jungers et al. [114] determined that acetone afforded extracts of ambient air particulate samples exhibiting the greatest mutagenicity. Similarly, Krishna et al. [115] found that acetone was more effective than dichloromethane for extraction of mutagens from air particulate samples collected using a Hi-Vol sampler; cyclohexane was the least-effective solvent, and Lo¨froth et al. [116] found that acetone afforded the most efficient extraction of air particulate sampled by electrostatic precipitation. Preidecker [117] found a mixture of benzene, methanol, and dichloromethane to be more effective than cyclohexane for extraction of mutagenic activity from air particulate collected in Houston, Texas. Morin et al. [59] compared three different solvents (dichloromethane, methanol, and acetone) for their efficiencies in the extraction of compounds from sidestream cigarette smoke; acetone extraction produced the highest levels of mutagenic activity. However, Jungers et al. [114] cautioned that while acetone potentially afforded greatest extraction efficiency for some samples, its use also resulted in co-extraction of inorganic salts; therefore, they recommended use of dichloromethane. DMSO, although directly compatible with the Salmonella assay, was found to be the least effective extraction solvent. Montreuil et al. [118] found that dichloromethane was the most effective solvent for extraction of mutagens from fresh diesel particulate; dichloromethane alone extracted 97% of the mutagenic activity recovered using sequential extraction by dichloromethane, methanol, acetone, and acetonitrile. However, the authors also noted that weathered particulate samples may contain a higher proportion of polar organic material; in this case, acetone may be more effective than dichloromethane. Nielson [119] compared dichloromethane and acetone extractions using particulate samples from the International Programme on Chemical Safety (IPCS) study designed to investigate intra- and inter-laboratory variations associated with preparation and bioassay of complex environmental mixtures [120]. He determined that ancillary Soxhlet extraction of urban particulate material with acetone following two extractions with dichloromethane resulted in extraction of substantial additional mutagenic activity; these results were based on bioassays using the TA98 and TA98NR Salmonella strains. In contrast, secondary acetone extraction of diesel particulate resulted in extraction of minimal additional activity. Nielson also reported that dichloromethane extracts were better suited to fractionation and chemical analysis, and is the best choice as a general solvent for extraction of complex environmental mixtures. May et al. [6] reported that dichloromethane was the most widely used extraction solvent for studies of standard reference materials SRM 1649 (urban air particulate) and SRM 1650 (diesel particulate). Buschini et al. [90] found both toluene and acetone extracts of air particulate from Parma, Italy, to be genotoxic in three different short-term bioassays, including the Salmonella assay. However, there were different responses among the three assays to the two extracts, indicating differences in the compounds extracted by each solvent. The different genotoxic

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responses were attributed to the ability of toluene to extract moderately polar and neutral compounds, while acetone extracted more polar compounds. Toluene extracts were also found to be cytotoxic in the Salmonella assay. Williams et al. [121] compared methanol, dichloromethane, toluene/ethanol (3:1 v/v), and acetone for extraction of air particulate impacted by wood smoke. They reported possible synergistic or degradation effects when toluene/ethanol and acetone were used. Methanol and dichloromethane both exhibited satisfactory extraction of mutagenicity, but methanol extracted the greatest amount of organic material and dichloromethane the least of all the solvents tested. The authors recommended use of dichloromethane over methanol as an extraction solvent for wood smoke-impacted particulates because of its greater volatility. Multiple solvent systems, used sequentially or in combination, also have been commonly used for extraction of air particulate. The most popular choices of multiple solvent sequential extraction combinations are sequential extraction with dichloromethane then acetone [122–127], and sequential extraction with dichloromethane then methanol [18,128–132]. Numerous other examples of sequential extraction combinations include cyclohexane and dichloromethane [133], sequential extraction with methanol followed by cyclohexane [134], sequential extraction with toluene followed by methanol [135], sequential extraction with dichloromethane followed by acetonitrile [136], sequential extraction with dichloromethane followed by hexane [137], sequential extraction using methanol, acetone, chloroform and cyclohexane [138], extraction in diethylether/cyclohexane (8:2 v/v) [62], and 1:1:1 tri-solvent systems of methanol/dichloromethane/ toluene [139], and methanol/benzene/dichloromethane [140]. Multi-solvent extraction systems are used in order to maximize the amount of extracted organic material from a variety of matrices. Some studies report that sequential extraction with solvents of differing polarity (semi-polar and polar) is required to achieve maximum effective extraction of mutagenic material [129]. Viau et al. [141] assessed both single- and multiplesolvent systems through calculation of mutagenic potential (Salmonella assay) of extracts of air particulate material sampled using a Hi-Vol in Lexington, Kentucky, USA. Mutagenic potential was calculated as extract yield in milligrams multiplied by revertants per milligram of extract. The authors determined that each of the solvent systems studied (benzene followed by acetone; benzene; acetone; benzene followed by methanol) afforded similar mutagenic potentials. However, sequential extraction by benzene followed by acetone afforded extracts containing fewer non-mutagenic co-extractives. The authors also concluded that benzene and acetone individually extracted different classes of mutagens. Krishna et al. [115] concluded that sequential extraction with acetone followed by dichloromethane afforded greater mutagenic response than acetone alone. Walker [138] used four individual solvents sequentially after determining a single solvent was not sufficient for adequately extracting the various

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classes of compounds to be examined in their epidemiological study of Houston air. McCalla et al. [129] determined that methanol was the most effective solvent for steel foundry air particulate, but subsequent extraction with dichloromethane resulted in a 5– 10% increase in total observed mutagenic activity. Dichloromethane alone extracted less than 50% of the mutagenic activity. These results indicated greater solubility of the organic air particulate matrix in methanol. De Martinis et al. [125] found that sequential Soxhlet extraction in dichloromethane followed by acetone yielded 20 and 10%, respectively, of the total mass of air particulate from Sa˜o Paulo, Brazil. The dichloromethane extract contained nonpolar and moderately polar organic compounds, while the acetone extracted more polar organic compounds and some inorganic species. Claxton et al. [22] dramatically illustrated the differences in percent mass recovery and mutagenicity of sequential dichloromethane and acetone extracts using data from De Martinis et al. [125], and showed that approximately one-third of the organics by weight could not be extracted using dichloromethane exclusively. Gundel et al. [142] used sequential extraction with cyclohexane, dichloromethane, and acetone for samples of inhalable air particulate collected in Elizabeth, NJ. Volume reduction of the composite solvent extracts afforded primarily acetone extracts that were subsequently fractionated using a cyanopropyl stationary phase as described below. The authors concluded that sequential extraction processes are more effective in dissolving compounds strongly adsorbed to the particulate matrix; these compounds become more soluble in solvents of lower polarity once they are desorbed from the matrix. Similarly, McCalla et al. [129] concluded that matrices such as steel foundry dust with a predominance of particles that have active surfaces (e.g., silica and iron oxides) may require a polar solvent such as methanol for extraction in addition to initial extraction in a nonpolar solvent, but the resulting extracted material may be entirely soluble in a nonpolar solvent such as dichloromethane. 2.2. Extraction method Hermann [143] estimated that 20% of the variation in mutagenic activity of complex mixtures is attributable to the extraction process. Soxhlet extraction has been the most common extraction method [40,43,44,46–49,51–53,55,57– 60,65–72,74,75,77,80,86,87,89–91,96,98,99,118,123,125,132, 134–136,144–147], followed by sonication [4,6,19,29,31,32, 36,45,50,56,60,62,63,73,94,106–110,133,139,140,148,149]. Shaking of filters immersed within the solvent system [38,82,92] has also been conducted. Barale et al. [84] reported that performing sonication and Soxhlet extraction using acetone yielded 20–30% more mutagenic matter than using either single-extraction method. However, other investigators [150] have shown that artifact formation is more likely with Soxhlet extraction, but the efficiencies of sonication and Soxhlet extraction are equivalent [151–153]. Van Houdt et al. [153] compared

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extraction of mutagens from both indoor and outdoor particles by Soxhlet and sonication in methanol, and found both methods to be satisfactory. The authors also performed secondary extractions using newborn calf serum and lung lavage fluids from pigs in order to simulate human exposure via inhalation. However, Krishna et al. [154] reported that sonication was superior to Soxhlet extraction for mutagenicity studies of airborne particles. Williams et al. [121] recommended use of sonication over Soxhlet extraction for air particulate impacted by wood smoke as due to the potential for compositional and/or mutagenicity changes when using the Soxhlet method. As part of their report on the IPCS study, Claxton et al. [120] reported that mutagenic activity in Soxhlet-based studies was more variable than studies employing sonication as an extraction method. Morin et al. [59] compared Soxhlet and ultrasonication for extraction of sidestream cigarette smoke particles; ultrasonication produced the most consistent results. However, Carvalho et al. [155] reported that ultrasonication compromised the integrity, and therefore potentially the corresponding mutagenic activity, of extracts of airborne particulate samples as a result of formation of new ionic compounds. For Soxhlet extraction, volume of solvent used, solvent cycling times, extraction times and thimble volumes, may all influence extraction efficiency of mutagenic activity [120]. Correspondingly, solvent volume, sonication time, number of sonication cycles, and temperature can influence extraction of mutagens using sonication [120]. Although the aforementioned methods are representative of those reported in the bulk of the literature, other extraction techniques and/or solvent systems have been investigated. Krishna et al. [154] found that soaking with acetone for 30 min afforded the highest mutagenic activity from air particulate sampled using a Hi-Vol, and thus offered a simple and efficient extraction procedure. The effectiveness of the acetone-soaking method was attributed to loose adsorption of mutagens with the particles coupled with high solubility of mutagens in acetone. More recently, Kuba´tova´ et al. [156] used hot pressurized water for both extraction and rudimentary fractionation of wood smoke and diesel exhaust particulate; genotoxicity was measured using the SOS Chromotest. Extraction was performed by hot pressurized water delivered to a supercritical fluid extraction cell using a syringe pump. The extraction cell was mounted in the oven of a gas chromatograph. Extraction of analytes according to polarity was accomplished by sequential 30-min extractions at varying temperatures ranging from 25 to 300 8C; increasing temperature resulted in extraction of decreasingly polar compound classes. The authors reported this method to be effective for compounds extracted by multiple organic solvent systems, in addition to being highly effective for extraction of more water-soluble polar compounds. Accelerated solvent extraction (ASE) is a recently developed extraction method that is finding rapidly increasing numbers of applications in environmental analytical laboratories globally. Using this method, the volume of solvent, solvent cycling time, extraction time, pressure, and solvent

temperature can all be programmed into the instrument. This new technology is now finding application in investigations of mutagenicity of airborne particulate [95,97]. 2.3. Fractionation based on solvent extraction Savard et al. [149] used extraction in a series of five solvents not only as a means of extraction, but also in an attempt to partition extract components into discreet fractions. Sonication in hexane, hexane:diethyl ether (9:1 v/v), hexane:diethyl ether (1:1 v/v), diethyl ether, and methanol yielded five fractions that showed trends in increasing/decreasing mutagenicity with polarity. However, the method was ineffective in isolating mutagenic activity in discreet fractions. Jungers and Lewtas [157] assessed mutagenic yield from air particulate by sonication and Soxhlet extraction using six solvents (cyclohexane, dichloromethane, acetone, methanol, toluene, and dimethyl sulfoxide); Soxhlet extraction using dichloromethane efficiently removed mutagens from the samples. Daisey et al. [158] used sequential extraction by cyclohexane, dichloromethane, and acetone to extract air particulate from New York City into nonpolar, moderately polar, and polar fractions, respectively. Pellizzari et al. [159] developed a scheme based on initial extraction of ambient air particulate in cyclohexane by sonciation, followed by extraction of the particulate in methanol. Møller and Alfheim [160] studied the potential for fractionation of toxic and mutagenic compounds based on sequential extraction of coal combustor particulates using solvents of increasing polarity; two particle-size fractions were sequentially extracted with pentane, benzene, and ether. The sum of mutagenic activities of the individual solvent extracts produced using this extraction scheme were compared with an extract prepared by acetone extraction; the acetone extract exhibited a higher mutagenic activity than the sum of the activities from the sequential extractions. The authors also observed the mutagenic activity to be spread across all three extracts prepared by the sequential extractions; therefore, mutagenic activities were not effectively separated and isolated. Butler et al. [144] extracted air particulate sequentially in cyclohexane, dichloromethane, and acetone to provide initial separation of extractables into nonpolar, moderately polar, and polar constituents, respectively. Ducatti and Vargas [148] sequentially extracted air particulate from Porto Allegre, Brazil, with cyclohexane (nonpolar fraction) and dichloromethane (moderately polar fraction) by sonication. Using the nitroreductase and O-acetyltransferase-deficient strains TA98NR and TA98/1.8DNP6, the authors were able to determine that nitro-PAHs were primary contributors to mutagenicity, but the use of the two extraction solvents separately did not appear to offer any advantage in terms of assessing genotoxic response of the samples. May et al. [6] used a sequential solvent partitioning–silica column procedure for fractionation of extracts of SRM 1649 (urban air particulate) and SRM 1650 (diesel particulate). In

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contrast to previous methods based on solvent partitioning, this method resulted in mass and mutagenicity recoveries in excess of 80% for both matrices. Dichloromethane extracts of airborne particulate were partitioned into hexane; the soluble hexane fraction was loaded onto a silica SPE column and subsequently separated into three subfractions through elution with hexane, dichloromethane, and methanol/dichloromethane. The insoluble hexane fraction and the residual particulate from the dichloromethane extraction were both separately extracted with methanol. Analysis of the hexane subfractions of both diesel and urban air particulate determined the presence of nonpolar compounds including aliphatics, substituted benzenes, and PAHs. The dichloromethane subfractions contained moderately polar organics including nitro-PAHs, dinitro-PAHs, PAH-ketones, PAH-aldehydes, quinines, and anydrides. The methanol/dichloromethane subfractions contained polar organic compounds including heterocyclics, PAH-acids, aliphatic acids, alcohols, esters, dintroarenes, and oxygenated nitro-PAHs. For urban air, the dichloromethane and methanol/dichloromethane subfractions contained 6.8% and 26.2% of the total extract mass, and 17.8% and 51.8% of the total mutagenicity, respectively. For diesel particulate, these subfractions contained 2.6% and 37.4% of the total extract mass, and 35.5% and 41.3% of the total mutagenicity, respectively. Gundel et al. [146] developed a very simple fractionation procedure based on solubility for the investigation of mutagenicity associated with polar compounds in airborne particulate. Particulate samples were Soxhlet-extracted sequentially with cyclohexane, dichloromethane, and acetone. The acetone extracts were evaporated to dryness, and the residue sonicated in dichloromethane (polar fraction). The dichloromethane-insoluble fraction was then sonicated in acetone, affording a residue and acetone-soluble fraction (very polar fraction). The polar and very polar subfractions were subjected to chemical-class testing and Salmonella bioassays. Numerous oxygenated compound classes were identified included carboxylic acids, aldehydes, ketones, alcohols, and phenols. Infrared spectra indicated the presence of nitro compounds, organic nitrates or nitrites, amines, and amides. Bioassays were conducted using TA98, TA98NR, and TA98DNP6 strains. The acetone-soluble compounds from SRM 1649 accounted for 36% and 40% of the direct and indirect TA98 mutagenic activities, respectively. The authors noted the significant contribution of acetone-soluble compounds to mutagenic activities of airborne particulate, and identified the need for improved fractionation schemes for polar organics in order to perform more detailed bioassaydirected fractionation studies.

3. Fractionation A myriad of fractionation schemes have been developed for studies of mutagenicity of air particulate material. For the purposes of this review, many of these methods are discussed according to three general

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schemas for the processing of crude organic solvent extracts from air particulate samples: (1) acid/base/ neutral partitioning followed by fractionation using open-column chromatography (silica/alumina) and/or HPLC; (2) fractionation based on NP-HPLC using a cyanopropyl or chemically similar stationary phase; and (3) fractionation by open-column chromatography followed by NP-HPLC using a cyanopropyl or similar stationary phase. In these cases, the HPLC methods may be preparative, semi-preparative, or analytical scale in nature, and can represent preparative and/or Level 1 and higher fractionations. Variations based on acid/base/ neutral partitioning followed by a chromatographic separation result in fractionation based both on acid/ base/neutral and polarity characteristics, while the chromatographic methods result in separations based exclusively on polarity. Although some of the methodologies used in contemporary studies of mutagenicity of air particulate do not represent significant advances in technology over the past 30 years, their simplicity, low cost, effectiveness, and robustness combine to result in their continued application in modern laboratories. 3.1. Acid/base/neutral partitioning followed by fractionation There is an abundance of early reports on the mutagenicity of air particulate that relied on an initial fractionation based on acid/base/neutral partitioning. In an early study, Teranishi et al. [99] fractionated benzene extracts of air particulate samples in Kobe, Japan, using an acid/base/neutral fractionation procedure followed by further fractionation of the neutral fraction on a silica column. The silica column procedure afforded three fractions; aliphatic (hexane elution), aromatic (benzene elution), and oxygenated (ether elution). Reconstitution of the fractions resulted in 75% recovery of the mutagenic activity of the crude extract. The bulk of the mutagenic activity was detected in the acidic, aromatic, and oxygenated fractions. Manabe et al. [107] fractionated extracts of diesel particulate into diethyl ether-soluble neutral, acidic, and basic fractions. Through GC/MS analyses and use of synthesized standards, the authors determined the presence of nitroacetoxypyrenes and nitrohydroxypyrenes in the extracts. It was estimated that 1-nitro-3-acetoxypyrene and 1-nitro-3-hydroxypyrene accounted for 12% and 9% of direct TA98 activity, respectively. Kolber et al. [161] used an acid/base/neutral and solvent (dichloromethane, cyclohexane, and nitromethane) partitioning scheme based on a method

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reported by Lee et al. [71] to generate six fractions from extracts of ambient air particulate collected in five locations in the United States. The six chemical classes included acids, bases, PAHs, polar neutrals, nonpolar neutrals, and insolubles (cyclohexane). Typical mass recoveries were greater than 80% for the overall chemical fractionation procedure. Fractions were bioassayed in Salmonella and analyzed by GC/MS; compounds identified in the various fractions were compared with a list of known toxic chemicals. The authors found the limited mass of extracted material available for fractionation and analysis to be a chronic problem. Austin et al. [162] used the same initial acid/ base/neutral and solvent partitioning scheme, with additional fractionation of the polynuclear aromatic hydrocarbon (PNA) fraction (defined as the ‘‘PAH’’ fraction in Kolber et al.) using silica gel, for comparison of mutagenicity of diesel particulate, cigarette smoke condensate, coke oven emissions, and roofing tar. The PNA fraction accounted for 45% and 52% of the total direct- and indirect-acting activities, respectively. Two of the four fractions from the silica gel fractionation of the PNA fraction (1:1 hexane/dichloromethane and dichloromethane) also made primary contributions to total mutagenicity. Williams et al. [163] used a procedure similar to that of Tokiwa [77] for a comparative characterization of diesel particulates, coke oven emissions, roofing tar vapours, and cigarette smoke condensates; the acid/ base/neutral partitioning steps were followed by a silica gel column fractionation. This protocol yielded fractions including organic acids, organic bases, cyclohexane insoluble compounds, polar neutrals, nonpolar neutrals, and PACs. This method exhibited good mass recoveries (>80%) for all samples except the cigarette smoke condensate, which was attributed to incomplete extraction of organic acids. Pitts et al. [131] used an acid/base/neutral fractionation scheme in concert with Salmonella strains TA98 and TA98NR and GC–MS and HPLC analyses to identify 6-nitrobenzo[a]pyrene, 9-nitroanthracene, 1-nitropyrene, and 5H-phenanthro[4,5-bcd]pyran-5-one in diesel exhaust particulates. Matsumoto and Inoue [164] used acid/base/neutral fractionation, followed by a silica gel column separation, and additional fractionation of the moderately polar neutral fraction using RP-HPLC of extracts of air particulate from Osaka, Japan. The silica gel fractionation yielded only moderate recovery of direct-acting mutagenic activity. The mutagenic moderately polar neutral fraction (benzene elution of the silica column) was fractionated into 10 subfractions on a 150 mm  6 mm i.d. ODS column using an isocratic

acetonitrile/water (8:2 v/v) mobile phase. This procedure was not successful in focussing mutagenic activity in specific subfractions; rather the activity was more widely distributed throughout 5 of the 10 subfractions. Subsequent analyses of the subfractions using GC/MS identified a range of moderately polar compounds, including oxy-PAC and aza-arenes. Schuetzle and Lewtas [1], Nishioka et al. [165,166], and Lewtas et al. [46] have all used acid/base/neutral partitioning in fractionation schemes developed for ambient urban air particulate. Acid/base/neutral partitioning (preparative-scale fractionation) was followed by silica gel open-column chromatography (preparative-scale fractionation) that afforded five fractions resulting from elution with hexane, hexane:benzene, dichloromethane, methanol, and acidic methanol. The dichloromethane fractions from the silica fractionation were then subjected to Level 1 and Level 2 fractionations using NP-HPLC. In the application of this methodology to Philadelphia air particulate, Nishioka et al. [166] found the majority of both direct-acting ( S9 metabolism) and indirect-acting (+S9 metabolism) mutagenic activities in the dichloromethane and methanol fractions resulting from silica column chromatography of the neutral parent fraction; the dichloromethane fraction exhibited 23% and 45% of the S9 and +S9 activities, respectively, while the methanol fraction contained 29% and 21%, respectively. Subsequent Level 1 fractionation of the dichloromethane fraction using NP-HPLC on a 4.6 mm i.d. silica HPLC column with a methanol:dichloromethane:hexane gradient elution program resulted in recovery of 45% of the +S9 activity and 27% of the mass in a single fraction; this fraction was subjected to further fractionation (Level 2) using the same HPLC column and a dichloromethane:hexane gradient. This Level 2 fractionation resulted in isolation of two fractions exhibiting significant +S9 mutagenic activity; analysis of the least-polar of the two fractions using GC–MS techniques (in both EI and NCI modes) resulted in identification of a series of hydroxylated nitro aromatic and hydroxylated nitro PAC as primary mutagenic compounds. This study demonstrated the power of the coupling of the separation potential of HPLC and capillary GC with the qualitative abilities of mass spectrometry. However, tracking of recoveries of both mutagenicity and mass became increasingly difficult at higher levels of fractionation. Further investigation of the hydroxyl nitro-PAC-containing subfraction from the Level 2 fractionation was not possible due to the limitation of sample quantity; therefore, the observed mutagenic activities could not

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be unequivocally attributed to the nitro compounds identified. In addition, the entire Level 2 and Level 3 investigations focused on the dichloromethane fraction from the silica column fractionation, despite the detection of significant mutagenic activity in the more polar methanol fraction. Greenberg et al. [122] used a method slightly modified from Nishioka et al. [166] to study seasonal composites of dichloromethane extracts of air particulate collected in Newark, New Jersey. Level 1 and 2 fractionations were carried out using a semi-preparative (9.4 mm i.d.  250 mm) silica column with UV adsorption and fluorescence detection and hexane/ dichloromethane mobile phases. Level 1 fractionation afforded four fractions, while Level 2 fractionation yielded four fractions, all of which ranged in polarity from nonpolar to polar. Bioassays were carried out using TA98, TA100, TA98NR, and TA98DNP6 strains. Observations from this study are as follows: 1. The bulk of mutagenic activity was associated with strong alkylating agents more polar than PAHs and nitro-PAHs. 2. Profiles of mass and mutagenic activities were generally similar at the acid/base/neutral and opencolumn preparative fractionation stages. 3. Level 1 fractionation showed a shift toward more polar compounds in summer samples. 4. Polar compounds afforded by additional extraction by acetone exhibited increased mutagenic activities in TA100, compared to TA98, particularly in the acidic fractions. 5. Hydroxynitro-PAHs were not amenable to analysis by GC–MS; HPLC with diode array UV detection was preferred. 6. Hydrolysis of 2-acetoxy-1-nitronaphthalene showed potential for artifact formation in acid/base/neutral fractionation procedures. 7. 2-Nitro-6H-dibenzo[b,d]pyran-6-one and 4-nitro6H-dibenzo[b,d]pyran-6-one were detected in a polar fraction of the dichloromethane extracts; the 2isomer was estimated to be responsible for roughly 20% of total mutagenic activity. Wise et al. [15] used a combination of acid/base/ neutral partitioning, silica gel fractionation, and NPHPLC in a very detailed chemical characterization of a large (49 g) sample of Philadelphia air particulate; the mutagenic activity of this sample is described in a companion paper [167]. Silica gel was used to separate the neutral fraction into five fractions using solvents of increasing polarity; hexane, hexane/benzene (1:1 v/v),

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dichloromethane, methanol, acidic methanol. The silica gel fractionation afforded 99% mass recovery. The aliphatic (hexane) and acidic polar (acidic methanol) fractions exhibited little or no mutagenic activity, while the aromatic (hexane/benzene), moderately polar (dichloromethane) and highly polar (methanol) fractions accounted for 8.4%, 23%, and 29%, respectively, of the direct-acting (TA98) mutagenic activity and 12%, 45%, and 21%, respectively, of the indirect-acting (TA98 + S9) mutagenic activity. The PAC-rich hexane/benzene fraction was further fractionated using an aminosilane NP-HPLC column with a 2% dichloromethane/pentane mobile phase. This NP-HPLC procedure afforded eight fractions, and resulted in separation of PAHs based on number of aromatic rings. Alkyl derivatives of PAHs eluted in the same fraction as the parent compounds. All eight fractions were characterized using GC–MS which resulted in identification of a broad range of PACs and their corresponding alkyl derivatives. The isocratic elution program resulted in the separation requiring over 200 min to complete. In a separate procedure, the aromatic fraction was fractionated using a combination of NP-HPLC and SPE to afford fractions designated as aliphatic, PAH, polar I, and polar II; these fractions were analyzed for nitro-PAHs; eight compounds were reported. 1-Nitropyrene was effectively separated from the PAHs and eluted in the polar I fraction. However, compounds such as 9-nitroanthracene and 3-nitrofluoranthene eluted primarily in the PAH fraction. Wu et al. [123] used a method modified from those of Nishioka et al. [165,166] and Wise et al. [15] to study extracts of air particulate collected in Newark, New Jersey. This method was based on acid/base/neutral partitioning (Level 1) followed by open column silica gel chromatography (Level 2) and NP-HPLC (Level 3). The neutral fraction was fractionated into four fractions on the silica column using hexane (aliphatics), 1:1 hexane/benzene (PAHs and nitro-PAHs), dichloromethane (moderately polar), and methanol (highly polar). The primary mutagenic fractions (moderately polar and highly polar) were further fractionated using a semi-preparative silica column (9.4 mm i.d.  250 mm) with a methanol/dichloromethane/hexane gradient elution program that afforded four fractions. Analyses of the NP-HPLC fractions were carried out using GC/MS, FTIR, and HPLC techniques. Mass recoveries were greater than 60% from the partitioning procedure, and greater than 75% for the silica column procedure; recoveries of mutagenic activity were not reported. Recoveries of moderately polar compounds from NP-HPLC were roughly 80%, while recovery of highly polar compounds was lower at

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roughly 60% due to retention of compounds on the column. Bioassays of the Level 2 fractions from the silica column procedure using TA98, TA98NR, and TA98DNP6 strains indicated clean separation of PAHs, nitro-PAHs, and dintro-PAHs. Chemical analyses determined the presence of quinines, ketones, and aldehydes in most mutagenic Level 2 fractions. Lewtas et al. [46] evaluated the acid/base/neutral partitioning followed by silica gel open-column chromatography method in terms of reproducibility of mass distribution, reproducibility of mutagenic activity, and recovery and distribution of mutagenicity using SRM 1649. Recoveries of both organic mass and mutagenicity (TA98) exceeded 80%; in terms of mutagenic activities these recoveries represented significant improvements over earlier work using acid/ base/neutral fractionation. The high percent recovery of mutagenicity was attributed to a lack of any synergistic or antagonistic effects. The organic acid fraction contained 65% of the direct-acting activity, while the hexane/benzene, dichloromethane, and methanol fractions contained 6%, 14%, and 14%, respectively. The authors estimated that PAHs contributed a maximum of 8% to the indirect-acting mutagenic activity of the extract, while the mono-nitro-PAHs contributed a maximum of 6% to the direct-acting activity. Møller et al. [87] used an acid/base/neutral partition scheme to fractionate airborne particulate from Oslo, Norway, in order to investigate variation in mutagenicity in relation to traffic and air pollution parameters. The neutral fraction was subsequently loaded on a silica gel column and eluted with cyclohexane, benzene, and ether to afford a nonpolar, polar aromatic, and oxygenated aromatic fraction, respectively. A further level of fractionation was achieved by further separating the nonpolar fraction on a combined silica gel–alumina column. Sequential elution with two volumes of pentane and 30% ether in pentane afforded aliphatic, PAH, and ‘‘transient’’ aromatic fractions, respectively. The method resulted in post-fractionation recoveries of 30% and 48% of direct- and indirect-acting mutagenicities, respectively. Individual fractions were recombined to investigate the loss of activity; these results indicated loss of mutagens during fractionation, as opposed to losses of any synergistic interactions. Similarly, Møller and Alfheim [160] found that roughly 80% of direct-acting mutagenicity was lost during acid/ base fractionation of particulates from a coal combustor; this effect was attributed to degradation of mutagens in the acid/base treatment. Alfheim et al. [81] later applied the same methodology to air particulate samples from Sweden and Norway.

A second procedure was also applied involving separation using NP-HPLC (silica). A hexane–dichloromethane–acetonitrile gradient elution program. Four NP-HPLC fractions were collected. Fraction 1 contained nonpolar compounds including alkanes, PAHs and alkylPAHs, and thia-PAHs; fraction 2 contained nitro-PAHs; fraction 3 contained ketones, aldehydes, quinones, and phenols; fraction 4 contained polar compounds and azaPAHs. Similar to the previous study [87], there were poor recoveries of mutagenic activity using the acid/base procedure; the authors speculated that mutagens may have been degraded through exposure to acidic or basic conditions. Recoveries of both direct and indirect mutagenic activities from the NP-HPLC fractionation procedure were practically complete. The value of this study in relation to analytical methods lies in the direct comparison of the efficacy of acid/base partitioning vs. NP-HPLC fractionation procedure in terms of recovery of mutagenic activity. Chan et al. [43] used a solvent partition scheme to fractionate extracts of diesel particulate into nine fractions designated as water-soluble, strong and weak acids, base and base salt, neutral polar, and two neutral nonpolar fractions; the neutral polar and neutral nonpolar fractions exhibited greatest mutagenicity in the Salmonella assay (TA98). Other applications of this general schema include work Dobia´sˇ et al. [45] on ambient coke oven air using acid/base/neutral fractionation followed by NP-HPLC (silica) and bioassay using TA98, TA100, and YG1041 (O-acetyltransferase and nitroreductase enriched) strains. A number of reports appear in the literature assessing genotoxicity of air particulate material collected in the Czech Republic; these studies used methods based on ˇ erna´ et al. [49] separated that of Lewtas et al. [46]. C dichloromethane extracts of PM10 particulate from the Czech Republic using acid–base portioning (based on the method of Lewtas et al. [46]) followed by further fractionation of the neutral fraction using silica gel chromatography into five subfractions. The highest mutagenicities (TA98 and YG1041) were attributed to nitro-PAHs in slightly polar subfractions. The major classes of compounds identified in the subfractions of the neutral fraction were aliphatic—aliphatic hydrocarbons; aromatic—PAHs and alkyl derivatives; slightly polar—nitro-PAHs, aromatic aldehydes, aromatic nitriles, aromatic diones, phthalic acid esters; moderately polar—aliphatic alcohols, aromatic ketones, phthalic acid esters; highly polar—carboxylic acid esters, alkoxy alcohols. ˇ erna´ et al. Topinka et al. [67] used the method of C [49] to study the formation of DNA adducts in

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mammalian cells by extracts of air particulate from the Czech Republic. The highest DNA adduct levels, comprising 75–90% of total adducts, were found in the aromatic and slightly polar subfractions containing PAHs and nitro-PAHs, respectively. Binkova´ et al. [66] used this methodology for comparison genotoxicity and embryotoxicity of air particulate from the Czech Republic; the aromatic subfractions of the neutral fractions showed the highest activities in both bioassays used, which was attributed to PAHs and alkyl their alkyl derivatives. The presence of these compounds was subsequently confirmed using GC–MS. Roughly 50% of DNA adducts formed during S9 metabolic activation were tentatively identified using HPLC. Binkova´ et al. [57] also used the acid/base partitioning method of Lewtas et al. [46] in a study of genotoxicity of coke oven and urban air particulate from the Czech and Slovak Republics. Similar to the scheme of Cˇerna´ et al. [49], the neutral fraction was further fractionated using silica gel chromatography into five subfractions (aliphatic, aromatic, slightly polar, moderately polar, highly polar) by sequential elution with solvents ranging in polarity from hexane to methanol. Genotoxicity of resulting fractions was assessed using in vitro acellular assays and TLC/HPLC analysis of 32P-postlabeled DNA adducts. For both coke oven and air particulate samples, the highest levels of DNA adducts were found in the neutral-aromatic fractions, followed by the neutral-slightly polar and acidic fractions. Zhao et al. [50] applied similar methodology to urban air particulate from Shanghai, China. The neutral fraction from the acid–base partitioning was fractionated on an XAD-2 resin–silica gel (1:1) column; sequential elution with hexane, benzene, and methanol afforded aliphatic, aromatic, and polar fractions, respectively. The highest mutagenic activities were associated with the acid, aromatic, and polar fractions from summer samples. Additional levels of fractionation were not undertaken to further isolate the observed mutagenic activities. 3.2. Normal-phase HPLC fractionation A second major iteration of common fractionation scheme involves the use of a NP stationary phase, typically cyanopropyl in more recent studies, to process extracts into compounds classes according to polarity. Some studies have employed fractionations using simple open-column methods on silica or alumina to perform rudimentary fractionations of air particulate. Thilly et al. [168] described a silica column fractionation procedure for diesel particulate extracts using silica

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and solvents of increasing polarity (pentane, pentane/ toluene, toluene, dichloromethane, methanol). This study was an early example of a NP fractionation used without prior acid/base/neutral partitioning. This method resulted in 70–80% mass recovery and virtually complete recovery of mutagenic activity. The authors concluded that 40–50% of mutagenicity of the nonpolar pentane/toluene fraction was attributable to fluoranthene and monomethyl phenanthrenes. Rivedal et al. [48] fractionated diesel exhaust particulates using silica into five fractions containing aliphatic hydrocarbons, PAHs, nitro-PAHs, dintroPAHs, and polar compounds. Highest mutagenic activities in the Salmonella assay (TA98, TA100, TA100NR, TA98NR, and TA98/1,8DNP6) were associated with the nitro-PAH and dinitro-PAH fractions. Pyysalo et al. [89] fractionated acetone extracts of air particulate from an industrial town in Finland on a deactivated silica column; elution with solvents of increasing polarity afforded five fractions. Mutagenic activities in these extracts were low, but detection of indirect-acting mutagenicity allowed the authors to conclude that compounds other than PAHs were responsible for the observed activity. Tuominen et al. [93] used the methodology of Pyysalo et al. [89] to investigate the genotoxicity of particulate and vapour phases of ambient air collected at three locations in Finland. In general, the highest genotoxicity was exhibited by the most polar fractions. Use of the Salmonella TA98NR strain indicated the presence of nitro-PAH as primary contributors to mutagenicity of the extracts. Hayakawa et al. [108] separated extracts of diesel particulate into five fractions on a silica gel column; over 60% of the total mutagenic activity was eluted in the dichloromethane fraction. This study was undertaken to assess the mutagenic contributions of nitro-PAH including 1-nitropyrene, and the 1,3-, 1,6-, and 1,8-dinitropyrenes; analyses were carried out by RP-HPLC with chemiluminescence detection. Sciherer-Roetman et al. [135] discouraged the use of both alumina and silica gel for fractionation of air particulate extracts because of low recoveries of mutagenicity, and recommended XAD-2 for ease of use and superior recoveries of both mass and mutagenicity. Comparative studies were carried out using toluene and methanol extracts of air particulate collected at rural and industrial sites in the Netherlands. Extracts applied to these stationary phases were fractionated by elution with solvents of increasing polarity. The authors found alumina to be superior to silica gel for separation of alkanes and PAHs. Interestingly, they were unable to recover spiked PACs (e.g., benzo[e]pyrene) from

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alumina and silica gel columns; these compounds have exhibited satisfactory recoveries in many other studies using these stationary phases. Rappaport et al. [136] fractionated dichloromethane and acetonitrile extracts of diesel exhaust particulate using a silica gel column (25 cm  1.5 cm i.d.) with solvent delivered using an LC pump. A rotary valve was used to enable the pump to deliver solvents of increasing polarity (hexane, dichloromethane, methanol) sequentially to the silica column. The dichloromethane fractions, exhibiting the highest mutagenic activities, were then subjected to two additional levels of fractionation using the same column, but with different solvents. The most active subfraction from the Level 3 fractionation was then further fractionated into three subfractions by HPLC using sequential isocratic elution with dichloromethane, acetonitrile, and methanol. The methanol subfraction was then further fractionated using a methanol/acetonitrile gradient elution program. The most active subfraction resulting from the two levels of HPLC separation was then characterized by mass spectrometry; the source temperature was increased over time to provide a level of selective sublimation of compounds. Ultimately, pyrene-3,4-dicarboxylic acid anhydride, was isolated and identified in the extracts. The presence of this weak direct-acting mutagen was confirmed through synthesis of an authentic standard. Although the compound identified was a minor contributor to the overall mutagenic activity of diesel particulate extract, this study is an early example of the effectiveness of successive levels of fractionation in a bioassay-directed study. However, the authors also noted that subfractions of diesel extracts were extremely complex and contained an ‘‘extraordinary assortment of organic adsorbates’’. In addition, this study emphasizes the need for large quantities of organic extracts in order to conduct thorough biological and chemical characterizations of air particulate. In this study, 40 g of diesel particulate afforded roughly 1 g of organic material in the dichloromethane extract and 750 mg in the acetonitrile extract. In turn, these extracts resulted in isolation of only 10–50 mg of individual compounds. Harger et al. [75] fractionated extracts of both the vapor and particulate phases of air sampled in Claremont, California, into nine subfractions using a semi-preparative silica HPLC column (10 mm i.d.  250 mm, 5 mm packing). The mutagenicity profile of the particulate extracts showed some activity to be associated with nitro-PAHs. However, the bulk of the mutagenic activity was found to be associated with compounds more polar than nitro-PAHs

Schuetzle et al. [19] used multi-level NP-HPLC and RP-HPLC fractionation in an exhaustive study of PAH derivatives in mutagenic fractions of diesel particulate extracts. The first level of fractionation of dichloromethane extracts was performed using silica columns using a hexane/dichloromethane gradient elution program; individual sequential pulses of acetonitrile and methanol resulted in elution of peaks corresponding to polar compounds. This procedure resulted in separation of the diesel extracts into nonpolar, moderately polar (designated as ‘‘transition’’ compounds), and polar compounds. There was some separation according to compound class among these three fractions. The nonpolar compounds were separated into two primary peaks consisting of 2–4-ring PAHs and 4–6-ring PAHs; the moderately polar compounds were separated into three peaks including high-molecular-weight PAH (6–8 rings), hydroxyl benzenes and 2-ring nitro-PAHs in the first peak, 2–6-ring nitro-PAHs, 3–4-ring hydroxy PAHs, and 3–4-ring PAH carboxaldehydes, 3-ring PAH quinones, and 2–3-ring PAH ketones in the second peak, and 3–5-ring PAH quinones, 5–7-ring hydroxyl PAHs, 3–4-ring PAH ketones, 3–5-ring dihydroxy PAHs, and 3-ring dinitro PAHs in the third peak; compounds eluting in the polar elution region included >6-ring PAH quinones, >3-ring PAH carboxylic acids, and azaaromatics. The moderately polar compounds were further fractionated using RP-HPLC and a methanol mobile phase to afford 30 individual subfractions for bioassays (TA98  S9). The bulk of both the indirectand direct-acting mutagenic activity was associated with the moderately polar compound fractions; 1-nitropyrene was responsible for roughly 30% of the direct-acting mutagenic activity of the total extract. Nitro-PAH in this study was analyzed by direct-probe high-resolution mass spectrometry (HRMS). The authors determined that greater than 95% mass and mutagenicity recovery was achieved using this HPLC method. They also questioned the origin of some oxy-PAH species that may have been derived from other oxy-PAH compounds during the analytical procedure. In a follow-up study, Schuetzle [13] used the method reported previously [19] to fractionate diesel particulate extracts. This study reiterated the importance of moderately polar compounds, including the nitro-PAH, as mutagens in diesel exhaust. As with the previous study, >90% mass and mutagenicity (TA98 S9) recoveries were achieved, and a series of oxy- and nitro-PAHs were identified using GC–MS and GC–MS–MS techniques. Mast et al. [110] used NP-HPLC with a semipreparative CN column and a hexane/acetone gradient to separate extracts of rice straw smoke into seven

C.H. Marvin, L.M. Hewitt / Mutation Research 636 (2007) 4–35

fractions; recoveries of mass and mutagenicity were >80% for all samples studied. The bulk of the mass and mutagenic activity was contained in fractions 2 through 5, which corresponded to elution of nonpolar to moderately polar compounds. Through analyses by GC–MS, alkylated PAHs were postulated as being responsible for the indirect-acting mutagenic activity in fraction 2, while dicarbonyl compounds were identified in fraction 4. Substituted phenols were prevalent in fraction 3, but at the time of the study had not been reported to be mutagenic. In a study similar to that of Helmig et al. [64] who identified nitrolactone compounds as mutagens in ambient air particulate extracts, Enya et al. [60] used preparative NP-HPLC fractionation with a hexane/ dichloromethane/acetonitrile gradient to fractionate extracts of diesel exhaust and air particulate collected in downtown Tokyo. Subsequent bioassays (YG1021 and YG1024) and GC–MS analysis resulted in identification of 3-nitrobenzanthrone as a powerful mutagen in these matrices. Ball and Young [65] used a 50 cm silica NP-HPLC column to fractionate diesel particulate extracts; this method resulted in 90% mass recovery, of which 75–80% was accounted for by aliphatics, and highly polar compounds including organic acids and esters. Chuang et al. [124] used two levels of NP-HPLC to investigate the mutagenicity of particulate material associated with indoor coal combustion in China. Dichloromethane extracts were fractionated using a semi-preparative (10 mm  300 mm) silica NP-HPLC column with a hexane/dichloromethane/acetonitrile/ methanol gradient elution program. Eight replicate injections resulted in fractionation of 60 mg of crude extract. This primary fractionation procedure afforded seven subfractions that were subsequently bioassayed in TA98 + S9. Mass recovery in this primary NP-HPLC fractionation was 97%. Most of the mass and mutagenicity was recovered in the second fraction that contained mostly PAH and alkyl derivatives as determined by GC–MS analyses. This PAH fraction contributed 43% of the organic mass and 61% of the total mutagenic activity of the crude extract. Based on these bioassay results, this PAH-rich fraction was subjected to another NP-HPLC separation using a semipreparative (9 mm i.d.  30 mm) aminosilane column with a hexane/dichloromethane gradient elution program. This secondary procedure afforded 11 subfractions roughly based on PAH molecular weight (i.e., number of rings). Bioassays of these subfractions showed the majority of mutagenic activity to be associated with alkyl derivatives of three- and

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four-ring PAHs. The authors also used GC–MS analyses to identify a wide range of compound classes associated with subfractions from both the primary and secondary NP-HPLC separations. The most polar fractions from the primary NP-HPLC fractionation accounted for roughly 30% of the total mutagenicity; major components of these subfractions included nitrogen and oxygen heterocyclic compounds. The cyanopropyl phase is reported to offer the greatest recovery of mutagens from combustion-related particulates [169]. In addition, the cyanopropyl phase is particularly suited to fractionation of extracts for bioassays, as the normal phase nature of cyanopropyl precludes the use of water or salts in mobile phases, which otherwise would have to be removed/evaporated prior to bioassays. Gundel et al. [142] developed a milligram-scale fractionation method for polar organics in acetone extracts of air particulate based on a cyanopropyl phase. These authors also reported that use of a stronger stationary phase, e.g., non-bonded silica, may result in irreversible adsorbtion of some polar analytes, much in the way these compounds can adhere to their parent particulate matrix. Similarly, in a study of the applicability of the cyanopropyl stationary phase for bioassay-directed fractionation studies of incinerator emissions, DeMarini et al. [63] reported that silica gel may irreversibly adsorb polar compounds that are abundant in incinerator emissions. This study also reported that the cyanopropyl stationary phase offers high mass and mutagenicity recoveries, the ability to tolerate wet extracts when used in NP mode, and equilibrates rapidly with mobile phases. ScihererRoetman et al. [135] discouraged the use of both alumina and silica gel for fractionation of air particulate extracts because of low recoveries of mutagenicity. Hannigan et al. [44] used a cyanoproyl SPE method to remove cytotoxic polar organic compounds from dichloromethane extracts of wood smoke prior to Salmonella bioassays. De Martinis et al. [125] separately fractionated dichloromethane and acetone extracts of airborne particulate from Sa˜o Paulo, Brazil on a cyanopropyl column; the dichloromethane extract exhibited roughly four-times greater mutagenicity than the acetone extract. Their results were similar to other studies in that S9-dependent activity was greatest in the nonpolar fractions containing PAHs, while S9-independent activity was associated with more polar compounds. Using GC–MS analysis, the most mutagenic HPLC fractions from the dichloromethane extract were found to contain ketones, aldehydes, and quinolinnes; the most mutagenic fraction from the acetone extract

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contained ketones, carboxylic acids, and aldehydes. Due to the complexity of the HPLC fractions, none of the compounds identified could be exclusively attributed to observed mutagenic activities. Hannigan et al. [170] and Durant et al. [171] have used elegant fractionation methods based on the cyanopropyl NP-HPLC stationary phase for the determination of human lymphoblast mutagens in extracts of urban air particulates from Los Angeles, CA and Washington, DC. In their study of southern California urban air, Hannigan et al. [170] initially separated the sample extracts into four primary subfractions of increasing polarity using a 10 mm i.d. cyanopropyl column and a solvent elution program beginning with 95% hexane/5% dichloromethane, followed by 100% dichloromethane, followed by 100% isopropyl alcohol. This procedure afforded two fractions containing nonpolar compounds (alkanes, low- to mid-molecular-weight PAC in the first fraction, high-molecular-weight PAC and some nitro-PAC in the second fraction), a semi-polar fraction (high-molecularweight nitro-PAC, keto-PAC, quinones), and a polar fraction (aldehydes, alcohols, acids). These four NPHPLC fractions were then subjected to a semipreparative size-exclusion chromatographic procedure to separate the aliphatic and aromatic compounds in each fraction. The use of size-exclusion chromatography is commonly used to separate aliphatic and aromatic compounds, as nonpolar aliphatics are largely inactive in the Ames assay. In addition, the presence of aliphatic compounds complicates the identification, by GC–MS or other methods, of aromatic compounds as potential mutagens in bioactive fractions or subfractions. The cutoff point between the aromatic and aliphatic fractions was determined by the elution time of 1,6-dinitropyrene. Subsequent to the size-exclusion chromatographic step, the semi-polar and polar fractions were further separated using the initial cyanopropyl column method, which afforded 10 subfractions from the semi-polar parent fraction and four subfractions from the polar parent fraction. Dose–response curves were generated for all fractions and subfractions, including the crude extract. All four fractions resulting from the initial cyanopropyl fractionation contained significant mutagenic activity; this mutagenic activity was manifested in four of the semi-polar aromatic subfractions and one of the semi-polar subfractions. The aliphatic subfractions of both the semipolar and polar fractions also contained significant mutagenic activity. However, despite application of this rigorous fractionation procedure that resulted in isolation of total sample mutagenicity in less complex subfractions, mutagenic

subfractions still contained multiple compounds and/or compound classes. The analytical method used for identification and quantitation was based on a list of target compounds that were judged to be likely present in a given fraction. Therefore, a shortfall of the overall approach was the presumption that the bulk any mutagenic activity detected could be ascribed to known and well-characterized compounds. In addition, this methodology is rather poorly suited to the determination of potentially mutagenic semi-polar or polar analytes; in the case of the Los Angeles air particulate sample, over half the total mutagenic activity could reside within the semi-polar or polar fractions. Compound identification was categorized as positive, probable, possible, and tentative, based on the degree of accuracy with which retention times and mass spectra correlated with those of authentic standards. The degree of association of the observed mutagenic activities with specific compounds was based on the concentration of a compound in the sample extract and mutagenic potency determined from bioassays of authentic standards. Using this method of calculation, cyclopenta[cd]pyrene was estimated to account for over 50% of mutagenic activity attributable to a single compound in Los Angeles air particulate. Other homocyclic PAH primary contributors included benzo[a]pyrene, benzo[ghi]perylene, benzo[b]fluoranthene, indeno[1,2,3-cd]pyrene, and benzo[k]fluoranthene. Significant semi-polar mutagens included 2-nitrofluoranthene and 6H-benzo[cd]pyren-6-one. A number of other compounds belonging to semi-polar and polar classes were identified using full-scan GC–MS, including aromatic ketones and quinones, and nitro-aromatics; none of these compounds could be linked to mutagenicity. In studies using methodology similar to that of Hannigan et al. [44,170], Durant et al. [171] used a variation of the above-described methodology for the determination of human lymphoblast mutagens in a Washington, DC, air particulate sample (SRM 1649), while Pederson et al. [172] studied airborne particulate from urban and rural sites in Massachusetts and upstate New York. The overall approach used by Durant et al. [171] was based on a four-level fractionation procedure based on polarity involving a preliminary cyanopropyl gravity column fractionation, followed by two semipreparative HPLC fractionations using a 10 mm i.d. cyanopropyl column and dichloromethane/hexane mobile phases, followed by a fourth fractionation step using size-exclusion chromatography. Thirteen PAHs were identified that accounted for roughly 15% of the total mutagenicity of the sample, including those compounds identified in the study by Hannigan et al.

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[170] outlined above. In addition, two high-molecularweight PAH (>278 amu) were identified as potent human lymphoblast mutagens, including naphtho[2,1a]pyrene and naphtha[2,3-a]pyrene. Although this protocol resulted in very good overall mass recovery (roughly 86%), recovery of mutagenicity was poor. The authors offered possible explanations including loss of mutagenic components during fractionation, or synergistic interactions between mutagens in the crude extract resulting in an artificially high activity for the bulk sample. Pederson et al. [172] used a single NP-HPLC separation on a 10 mm i.d. cyanopropyl NP-HPLC column to fractionate extracts of air particulate from the northeastern United States in order to investigate mutagenicity of PM2.5 samples on a regional basis. The NP-HPLC fractionation employing a three-step gradient elution program (95% hexane/5% dichloromethane, dichloromethane, 50% dichloromethane/50% isopropyl alcohol) afforded four fractions including two nonpolar fractions, a semi-polar fraction, and a polar fraction. Compound classes eluting in these fractions were as per those reported in the previous studies [44,170,171]. The greatest human-cell mutagenicities were detected in the semi-polar fractions, i.e., those fractions containing compounds frequently associated with mutagenicity of air particulate, e.g., nitro-PAH, keto- and aza-PAC. In this study, these fractions were not chemically characterized. 3.3. Open-column chromatography followed by normal-phase HPLC A third basic scheme involving fractionation of solvent extracts by open-column chromatography has been widely used in bioassay-directed fractionation studies of air particulate material. In many cases, the column cleanup was followed by further fractionation by NP-HPLC using a cyanopropyl or other bonded phase. These studies have targeted compound classes isolated according to polarity, e.g., PAC; the rationale behind this approach being that the majority of mutagens in air particulate are PAHs, PAH derivatives, or other PACs. Schuetzle et al. [13] developed methodology for the bioassay-directed investigations of polar PAC in air particulate samples based on the earlier work of Salmeen et al. [173]. This work was among the early studies that fully illustrated the concept of exploiting the resolving power of HPLC to identify chemical mutagens. Diesel particulate (NIST SRM 1650) was sequentially Soxhlet extracted in dichloromethane

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followed by methanol; the individual extracts were not composited. Subsequent bioassays showed that 96% of mutagenicity was associated with the dichloromethane extract. The dichloromethane extract was then subjected to fractionation using open column silica (5% deactivated silicilic acid) chromatography; elution by hexane afforded a fraction containing aliphatics and lower molecular weight PAHs. Based on bioassay analyses, the hexane fraction (22% by mass of extracted material) exhibited no mutagenicity. Subsequent elution of the silica column by methanol afforded a ‘‘moderately-polar’’ fraction containing primarily PAH derivatives and PAC. This fraction was further separated (Level 2 fractionation) by NP-HPLC on a 10 mm 30 cm  7.8 mm i.d. semi-preparative silica column. A series of eight subfractions were collected according to elution of a series of compounds spanning a polarity range from nonpolar, to moderately polar, to polar in nature. The moderately polar and polar subfractions contained 37% and 45% of the total mutagenicity, respectively. Subsequent bioassays showed that 79% of the mutagenicity of the parent fraction was recovered, the remainder being irreversibly adsorbed to the NPHPLC column. However, total mass recovery was much higher (98%). The authors also proposed the elution of specific individual compounds as common references in defining the separation of these three polarity classes in complex environmental mixtures. 1-nitronaphthalene was proposed as the end of elution of nonpolar compounds, while elution prior to, and after, 1,6pyrene quinone would constitute the moderately polar and polar elution windows, respectively. It does not appear that these compounds were universally adopted as elution standards. Subfractions classified as being moderately polar and polar were subjected to detailed chemical characterizations. Compounds identified in these subfractions using GC/MS included hydroxylPAHs and hydroxynitro-PAHs. One of the polar subfractions were subjected to a Level 3 fractionation by RP-HPLC (using semi-preparative C18 column with a water/acetonitrile gradient elution program), which was required to reduce the complexity of the subfraction. Subsequent bioassays of 1-min subfractions enabled prioritization for further chemical analyses. Plots of bioassay response (expressed as TA98 S9 revertants) vs. subfraction retention times resulted in an ‘‘Ames assay chromatogram’’, referred to earlier in this review as a mutation chromatogram. GC/MS analyses of the two subfractions exhibiting the greatest biological activities resulted in detection of 5–10 compounds in each; the components responsible for

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the observed activities were not identified. This study emphasized a recurring issue that continues to the present day; the problematic identification of polar mutagens. Braun et al. [128] fractionated dichloromethane extracts of particulates from residential oil burners on alumina columns using sequential elution of solvents of increasing polarity. Mass recovery for the alumina procedure was >70%. Mutagenic activity was contained primarily (>80%) in the benzene (nonpolar) or chloroform (moderately polar) fractions. Mutagenic fractions from the alumina procedure were further fractionated by NP-HPLC using a cyanopropyl column with a hexane/dichloromethane gradient elution program. Mutagenic activity was confined to the fraction known to contain nitro-aromatics. The authors reported that alumina was unsuitable for fractionation of extracts of hydrocarbon combustion, as 80% of mutagenic activity manifested through addition of metabolic activation was not recovered. They postulated that compounds may have been irreversibly adsorbed onto the alumina, and recommended use of cyanopropyl columns according to Lafleur et al. [169]. Both Legzdins et al. [18] and McCalla et al. [129] have conducted bioassay-directed fractionation studies of urban air particulate and steel foundry particulates, respectively. Methodologies used in these studies were based on multi-stage chromatographic protocols developed for isolation and determination of PACs in fuels and coal liquids [174–177]. Pooled composite extracts resulting from sequential 24-h Soxhlet extractions of the sample matrix using dichloromethane and methanol were fractionated using an open-column alumina procedure; the alumina column was eluted by solvents of increasing polarity (hexane–benzene–dichloromethane/ethanol (99:1 v/v)–methanol–methanol/water (4:1 v/v)). The hexane fraction contained primarily aliphatic compounds; the benzene and dichloromethane/ethanol fractions were pooled, as were the methanol and methanol/water fractions to afford nonpolar PAC and polar PAC fractions, respectively. The nonpolar PAC fraction was subjected to a sizeexclusion gel (Sephadex LH-20 polyhydroxylated dextran) chromatography to remove any residual aliphatics. In this case, the size-exclusion gel is employed as an adsorption column using a hexane:methanol:dichloromethane phase that promotes increased interaction of aromatic compounds with the stationary phase. As a result, material eluting prior to an elution time corresponding to naphthalene, is exclusively aliphatic in nature. Other analysts have used Sephadex LH-20 to effectively eliminate residual

aliphatics from air particulate extracts [19,178]. Subsequent Level 2 and Level 3 fractionations were carried out using NP-HPLC and RP-HPLC, respectively. The aforementioned methodology was used by McCalla et al. [129] for the isolation and identification of mutagens in samples of several hundred grams of fine steel foundry dust comprised of airborne particulates and binder emissions. The Level 2 NP-HPLC procedure was carried out on a preparative scale (25 cm  94 mm i.d. column) using a mixed aminocyano bonded phase (Whatman Partasil PAC) column with a hexane:dichloromethane:methanol gradient elution program. In a variation of the general schema, Casellas et al. [39] employed gel permeation chromatography (GPC) as an initial step to separate dichloromethane extracts of airborne particulate from Barcelona, Spain, into lipid, PAC and polar compound fractions; the lipid fraction contained over 85% of the mass of the crude extracts but less than 10% of the observed mutagenicity. The GPC procedure was performed to remove high-molecularweight biogenic and lipid compounds, some of which have been reported to exhibit antagonistic mutagenic effects as reported by Shah et al. [179]; these compounds can include those associated with airborne particulate including fatty acids [180]. The PAC fraction was then further separated using NP-HPLC (silica) followed by RP-HPLC. Key findings of this study included identification of over 80 compounds in the mutagenic fractions including aromatic ketones, quinines and aldehydes. In addition, the important contribution of nitroaromatic compounds was emphasized using nitroreductase-deficient Salmonella strains. Strandell et al. [52] applied an open-column silica fractionation procedure for a thorough chemical and biological characterization of ambient air, diesel, and gasoline engine particulate extracts. Five fractions were collected from the column based on sequential elution with hexane, hexane, 25% dichloromethane/hexane, dichloromethane, and methanol according to the method of Alsberg and Stenberg [53]. The dichloromethane fraction, which exhibited the greatest mutagenicity for all three extracts, was subjected to further fractionation using a NP-HPLC method with (10 mm  100 mm Lichrosorb DIOL column) a hexane/dichloromethane gradient elution program; this procedure afforded six subfractions for each extract; bioassays and analyses by GC techniques were conducted to compare the chemical and biological profiles of the three sample types. The most polar (latest eluting) of the subfractions contained the greatest mutagenicity for all three extracts; 51% for the gasoline

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engine, 39% for diesel, and 23% for air; this subfraction was found to be exceedingly complex and would have required further levels of fractionation to identify the compounds responsible for the observed mutagenicity. The authors also compared mutagenic activities of the conventional TA98 and nitroreductase-deficient TA98NR as measures of the importance of nitro-PAH to the biological activities of the extracts. Chemical analyses showed oxygenated PAC to be predominant in all subfractions of the dichloromethane fraction. Helmig et al. [64] used open column silica chromatography followed by Level 1 fractionation using NP-HPLC to fractionate dichloromethane extracts of ambient air particulate collected in California. Sequential elution with solvents of increasing polarity (pentane, dichloromethane, methanol) afforded fractions from the preparative silica column. Level 1 fractionation was performed using a semi-preparative silica HPLC column and afforded nine individual subfractions. Analysis of the most mutagenic (TA98 S9) of these subfractions by GC–MS resulted in identification of two mutagenic nitrodibenzopyranones, 2-nitro-6H-dibenzo[b,d]pyran6-one and 4-nitro-6H-dibenzo[b,d]pyran-6-one. Bioassays of authentic standards of the two compounds showed that the mutagenic activity in the most mutagenic of the NP-HPLC could be attributed to the 2-isomer, and that this compound was responsible for roughly 20% of the activity in crude extract. Many studies have employed HPLC, SPE, or opencolumn methods for Level 1 fractionations, without any subsequent separation procedures. In these cases, the resulting fractions are still exceedingly complex, and do not generally allow identification of single compounds or compound classes associated with mutagenic responses. Frequently, these methods serve to assist in assessment of mutagenic activities within the context of partial chemical compositions or other parameters. Alsberg and Stenberg [53] used an open-column silica column procedure to fractionate extracts of gasoline engine particulates; this paper is an early example of a very thorough chemical and biological characterization of a complex extract of particulate, and as a result is heavily cited in the literature. Five fractions were collected from the silica column based on sequential elution with hexane, hexane, 25% dichloromethane/hexane, dichloromethane, and methanol. Subsequent chemical analyses using GC methods identified roughly 20–40 compounds in fractions 2 through 4. The first hexane fraction (fraction 1) contained aliphatics, while the second hexane fraction (fraction 2) contained PAHs. The third fraction contained polyaromatic aldehydes, brominated and

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chlorinated derivatives of oxygenated-PAH, and oxygenated-PAH, the majority having one-to-five rings. Fraction 4 contained six-ring oxygenated PAH including polynuclear quinines and ketones; the most abundant compounds in this fraction were xanthone and phenalen-1-one. Fraction 5 contained polar oxygenated and nitrogenated compounds of relatively low molecular weights. The aliphatic fraction did not exhibit any response in the Salmonella assay, while fraction 2 (PAHs) showed the expected positive response with addition of oxidative metabolism. Fractions 3 and 4 exhibited the greatest mutagenic activities, while fraction 5 showed a lower response. The authors concluded that none of the predominant compounds identified in fractions 3 and 4 appeared responsible for the observed mutagenic activities; this paper did not report the presence of nitro-PAH or dinitro-PAH in the extracts, as in the case of later studies using the Alsberg et al. fractionation protocol [48]. In a companion paper, Alsberg et al. [181] used an additional Level 2 fractionation of the moderately polar fraction of gasoline particulate extracts using a 150 mm  11 mm i.d. NO2 HPLC column and an isocratic hexane/3% chloroform/0.1% ethanol mobile phase. Four subfractions were collected; the second subfraction 30% of the parent fraction TA100 S9 mutagenicity and approximately 80% of the TA100 + S9 mutagenic activity. Mass spectrometric data together with bioassays with a nitroreductasedeficient strain (TA100NR) indicated the presence of nitro compounds in the active subfractions. However, oxygenated PACs including 9H-fluoren-9-one, alkylfluorenones, and benzofluorenones were the most abundant compounds in the active subfractions. Ciganek et al. [51] used silica gel to fractionate air particulate extracts to investigate the association of mutagenicity with PAHs; sequential elution with hexane, hexane/dichloromethane, dichloromethane, and methanol afforded aliphatic, aromatic, semipolar, and polar fractions, respectively. Oda et al. [182] used an aminophase SPE method to fractionate extracts of particulate matter from Kimoto, Japan. The SPE cartridges were initially cleaned with acetone, and then conditioned with a series of solvents of decreasing polarity. Fractionation was achieved through sequential elution with hexane (aliphatics), cyclohexane (two to four ring PAHs), 20% dichloromethane/cyclohexane (large PAHs and nitroPAHs), dichloromethane (weakly polar), acetonitrile (moderately polar), and methanol (highly polar). Chorazy et al. [98] used a silica gel column fractionation procedure based on a method previously developed for characterization of coal liquids; this procedure was

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applied to investigation of the chemistry and mutagenicity of air particulate from Upper Silesia in southern Poland. Sequential elution with a series of solvents afforded a polarity-based fractionation according to solubility parameters. The first three fractions eluted sequentially with hexane, 15% benzene/hexane, and chloroform contained saturated hydrocarbons, PAHs, and polar aromatics and non-basic nitrogen, sulfur, and oxygen heterocyclics, respectively. More polar fractions were eluted with 10% diethyl ether/chloroform, 3% ethanol/diethyl ether, and methanol. The 15% benzene/ hexane through methanol fractions all exhibited mutagenic activity in the Salmonella assay; as expected, the PAH-containing fraction (10% diethyl ether/chloroform) exhibited increased activity in the presence of S9. Rivedal et al. [48] fractionated diesel exhaust particulate extracts on silica gel based on the methods used by Alsberg et al. [181] and Østby et al. [183]; sequential elution with cyclohexane, cyclohexane followed by 25% dichloromethane/cyclohexane, 25% dichloromethane/ cyclohexane followed by dichloromethane, dichloromethane, and methanol, afforded aliphatic, PAH, nitroPAH, dinitro-PAH, and polar fractions, respectively. The authors conducted bioassays using the conventional TA98 and TA100 Salmonella strains, and the corresponding nitroreductase and O-acetyltransferase-deficient strains, to determine the significant contributions of nitro-PAHs and dinitro-PAHs to mutagenicity of diesel extracts. DeMarini et al. [29] used an open-column silica fractionation procedure to investigate differences in the mutagenic activities of automobile and forklift exhaust particles. The column was eluted sequentially with hexane, 50% hexane/dichloromethane, dichloromethane, and methanol. Recoveries of extracted mass were 84% and 103% for the automobile and diesel extracts, respectively. The authors also found the distributions of mass to differ substantially between the two matrices; roughly 55% of the automobile extract mass eluted in the hexane fraction and 33% in the methanol fraction. In contrast, the corresponding masses for the diesel extract in these fractions were 29% and 58%, respectively. However, the sum of mutagenic activities (TA98 S9) of the individual fractions were roughly 4- and 14-fold higher than the crude extracts of the automobile and diesel particulate samples, respectively. 3.4. Ion-exchange chromatography Fractionation methods based on ion-exchange have been developed for matrices where solvent extracts can

be highly acidic, where the bulk of the mutagenic activity is attributable to polar compounds, e.g., wood smoke, and for which the methods described above have lacked effectiveness in terms of both extracted mass and mutagenicity recovery. Watts et al. [47] used a non-aqueous ion-exchange solid-phase extraction (SPE) procedure to fractionate particulate from municipal and hospital waste incinerators. As with wood smoke, extracts of these matrices can be highly acidic in nature and exhibit high degrees of cytotoxicity in the Salmonella assay. Dichloromethane extracts were loaded onto a quaternary ion-exchange resin and sequentially eluted with dichloromethane, methanol, methanol saturated with CO2, and 10% trifluoroacetic acid in methanol. The dichloromethane fraction, containing neutral compounds, contained 70% of the extracted mass, and did not exhibit any cytotoxicity in the Salmonella assay. Bell et al. [184] applied a similar procedure for bioassay-directed fractionation of particulates of wood smoke. The authors used sequential cation exchange followed by anion exchange. Extracts were loaded onto an Amberlyst 15 (sulfonic acid) cation-exchange column; elution with 20% methanol in dichloromethane yielded a neutral/acid fraction. The retained bases were eluted with 20% isopropylamine in methanol. The combined neutral/acid fraction from the cationexchange procedure was then applied to an Amberlyst 26 (quaternary amine) anion-exchange column; neutral compounds were eluted with dichloromethane. An acid fraction was eluted with CO2/methanol, and a polar acid fraction was eluted with 10% trifluoroacetic acid in methanol. Mass recovery using this procedure was reported as roughly 108%, with the base, neutral, and acid fractions accounting for 15%, 55%, and 35%, respectively. Thompson et al. [185] developed an anionexchange solid-phase extraction method for fractionation of extracts of ambient air and combustion source samples. Dichloromethane extracts of air particulate were loaded directly onto a 100–200 mesh AG MP-1 anion-exchange resin; the glass syringe resin column was then sequentially eluted with dichloromethane, methanol, CO2/methanol, and 5% trifluoroacetic acid in methanol. The resulting fractions were classified as neutral/basic, polar neutral/weak acid, weak acid, and strong acid. Highest mutagenic activities for two composite samples comprised primarily of wood smoke (78%) and a combination of wood smoke and mobile sources (51% wood smoke and 33% mobile sources), were contained in the dichloromethane fraction, despite the fact these fractions contained

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less than 30% of the mass of the extracts. Analysis of the fractions by GC–MS identified major compound classes: the dichloromethane fraction contained alkanes, aliphatic aldehydes, PAHs and alkyl PAHs, aromatic ketones, phthalate esters, aliphatic amides, alkoxy alcohols, and alkoxy phenoxy compounds; the methanol fraction contained phthalate esters, alkoxy alcohols, and aliphatic carboxylic acids; the CO2/ methanol fraction contained alkoxy alcohols, alkoxy phenoxy compounds, aliphatic carboxylic acids, and hydroxylated aromatics; the trifluoroacetic acid/ methanol fraction contained alkoxy alcohols, aliphatic carboxylic acids, methyl esters of aliphatic carboxylic acids, and methyl esters of aromatic carboxylic acids. Recoveries of mass and mutagenicity using this method were approximately 94% and 100%, respectively. DeMarini et al. [72] used an ion-exchange procedure modified from Bell et al. [184] and Thompson et al. [185] to fractionate extracts of air particulate collected in Boise, Idaho. This procedure afforded five fractions. The first three fractions were eluted according to the method of Thompson et al. [185]; the last two fractions were eluted using 2% trifluoroacetic acid in methanol and 10% trifluoroacetic acid in methanol, respectively. The neutral/base fraction (dichloromethane) contained roughly 80% of the mutagenic activity and 36% of the sample mass. Most of the observed mutagenic activity was attributed to PAHs. While these ion-exchange chromatographic methods have been demonstrated to be effective for these matrices it should be noted that ion-exchange resins can require considerable preparation, including pre-cleaning and conditioning. 3.5. Thin-layer chromatography Thin-layer chromatographic methods of fractionation have seen limited application to investigations of mutagenicity of air particulates. Applications of this technique were documented during the 1980s. Tokiwa [77] used an acid/base/neutral and solvent solubility fractionation method to process methanol extracts of air particulates samples from an industrial area. This procedure afforded ten fractions based on acid/base/ neutral character and solubility in diethyl ether, ethanol (designated as water soluble), methanol/water, cyclohexane, and nitromethane. These fractions were subjected to an additional level of fractionation using TLC followed by analysis by spectrophotometry or GC/ MS. Total recovery of mutagenic activity after fractionation was 58%.

25

Moriske and Ru¨den [62] used TLC to fractionate the neutral fraction from an acid/base/neutral fractionation of air particulate sampled in Berlin. The TLC procedure yielded three primary fractions containing aliphatics, PAHs, and polar neutral compounds. Bioassay with TA98 and TA98NR implicated nitro-PAHs as primary mutagens. Siak et al. [40] fractionated dichloromethane extracts of urban and suburban air particulate from southeast Michigan using thin-layer chromatography (TLC). The TLC fractionation resulted in 16 individual subfractions that were subsequently assayed in TA98, TA98NR, and TA98/DNP6. The bulk of the mutagenic activity (>80%) was observed in the most polar TLC fractions; compounds associated with these fractions exhibited reduced responses in the nitroreductase-deficient strains. TLC fractions containing the nitro- and dinitro-PAHs did not exhibit high levels of mutagenic activity (<3% of total mutagenicity). However, 1nitropyrene, 1,6-dinitropyrene, and 1,8-dinitropyrene were measured in the samples using HPLC after aqueous reduction to the corresponding amines. Butler et al. [144] developed a fractionation scheme based on different extraction solvents and followed by a TLC procedure as part of an ‘‘integrated chemical class/ bioassay system’’. Air particulate from several major urban centres from around the globe was extracted sequentially in cyclohexane, dichloromethane, and acetone. This extraction scheme was designed to provide initial separation of extractables into nonpolar, moderately polar, and polar constituents, respectively. Three different TLC mobile phases were used due to the varying nature of compounds in each of the three extraction solvents. In addition, various methods of detection were used to identify different compound classes on the TLC plates, e.g., ultraviolet absorption at 254 nm for PAHs, and potassium iodoplatinate reagent for aza-arenes. 3.6. Mutation chromatograms Conventional HPLC instruments are usually equipped with detection systems based on ultraviolet adsorption, fluorescence, or mass spectrometry. Successive levels of HPLC separation in bioassay-directed fractionation studies can result in large numbers of subfractions that are subsequently bioassayed. In this case, the bioassay essentially becomes another form of detection, with detector response based on mutagenicity rather than physical/chemical structure. Plotting the relationship of bioassay response with HPLC retention time affords a mutation chromatogram. Other common

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terminology used in defining this relationship includes bioassay chromatogram, Ames assay chromatogram, and mutagram. Examples of mutation chromatograms from Legzdins et al. [18] are shown in Fig. 3. Air particulate material from Hamilton, Ontario, Canada, was fractionated according to the protocol shown in Fig. 1. The nonpolar to moderately polar aromatic fraction (A23/ LH20) resulting from the alumina fractionation procedure was further separated by NP-HPLC using a semi-preparative polyaminocyano column. This NPHPLC procedure was modified from Wise et al. [14,15] and May et al. [16,17], which resulted in separation of PAHs according to benzologue class, in addition to effective and reproducible separation of more polar PACs. One-minute subfractions were collected from the NP-HPLC procedure and subjected to a multiple bioassay approach with a series of Salmonella strains (YG1021 S9, YG1024 S9, YG1029 + S9). Using this approach, 2-nitrofluoranthene, 1-nitropyrene, and 2-nitropyrene were confirmed by GC–MS as primary direct-acting mutagens (subfraction 31; Fig. 3), while benzo[a]pyrene and indeno[1,2,3-cd]pyrene were identified in subfractions (subfractions 21 and 24, respectively; Fig. 3) requiring metabolic activation to demonstrate mutagenic activity. Later work showed the fractions exhibiting high mutagenic activity in YG1024 (subfractions 45 and 47) contained dinitropyrenes; compounds responsible for the mutagenic responses in the most polar regions of the chromatograms (75–80 min) remain undetermined. Early examples of this methodology include Tokiwa et al. [56,109], Salmeen et al. [173,186], and Siak et al. [40]; these studies emphasized the importance of nitroand dinitro-PAHs as mutagens in a variety of air particulate matrices. Salmeen et al. [173,186] used NP-HPLC to generate mutation chromatograms for diesel particulates from both cars and trucks. Dichloromethane extracts were fractionated into 65 one-minute subfractions, and assayed with TA98 at a single dose per subfraction without metabolic activation. Polar material eluting later in the gradient elution program was collected as a single subfraction; this subfraction accounted for 17– 35% of the direct-acting mutagenic activity in the samples. Individual subfractions exhibiting greatest mutagenic activities eluted at retention times corresponding to standards of 1-nitropyrene and 1,8-dinitropyrene. The authors used the mutagenic activities derived from the active subfractions to estimate the concentrations of nitro- and dinitro-PAHs in the samples; 30–40% of the total recovered mutagenicity was attributed to six nitroarenes. However, the authors also stated that the

lack of dose–response data could result in significant uncertainties in the estimated mutagenic activities, and subsequently the concentrations of nitro- and dinitroPAHs, but the identification of these compounds as primary mutagens in the samples remained valid. Ball et al. [68] used the same methodology of Salmeen et al. [186] to investigate indirect-acting mutagenicity in diesel particulate extracts. The authors were unable to identify compounds associated with indirect-acting activity, but demonstrated the requirement for fractionation to reveal mutagenicity that would not otherwise have been detected in crude diesel extracts. Tokiwa et al. [56] used mutation chromatograms to illustrate the contribution of 1,6- and 1,8-dintropyrene to the mutagenicity of airborne particulate collected in Santiago, Chile. Following an initial fractionation on silica gel, the polar fraction was separated on a Zorbax ODS RP-HPLC column; 30-s subfractions were collected and assayed in TA98 S9. The bulk of the mutagenic activity was confined to a 2-min retention time window; subsequent bioassays of these active fractions with TA98/1,8DNP6, and analysis of the pooled active subfractions by HPLC using a Zorbax CN column, confirmed the presence of the dinitropyrenes. Tokiwa et al. [109] also produced an excellent study of the contribution of 1-nitropyrene and the dinitropyrenes to the mutagenicity of particulate material emitted from kerosene heaters and fuel gas and liquefied petroleum gas burners. Extracts were subjected to a Level 1 fractionation using a Sephadex LH-20 gel column; individual subfractions were assayed in TA97 S9 to afford a mutation chromatogram. The bulk of the mutagenic activity in the LH20 mutation chromatogram was confined to a relatively narrow retention time range. The active subfractions were pooled and subjected to a Level 2 fractionation using HPLC. Bioassay of individual 30-s subfractions showed the highest mutagenic activities corresponded to the dinitropyrenes and 1-nitropyrene; these compounds were estimated to account for 40–80% of the total mutagenic burden of the extracts. Subsequent analyses of the mutagenic subfractions by GC–MS confirmed the presence of these compounds. In another study, Tokiwa et al. [73] generated mutation chromatograms for a series of indoor and outdoor airborne particulates. The benzene fractions from a silica column procedure were separated on a RP-HPLC column and subfractions collected and subsequently bioassayed in TA98  S9. The active subfractions were pooled and analyzed by GC–MS. Direct-acting mutagens identified using this methodology included 9-fluorenone, 2-nitrofluorenone, 3,9- and 3,7-dinitrofluoranthenes, 1,6- and 1,3-dinitropyrene, 1-nitropyrene, and 3-nitrofluor-

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Fig. 3. Mutation chromatograms resulting from bioassays of 1-min subfractions from NP-HPLC separation of a nonpolar to moderately polar aromatic fraction from an urban air particulate extract. (A) The ultraviolet absorption profile (254 nm) and corresponding PAH molecular weight classes contained in each peak. (B–D) The net revertants from bioassays of 1-min subfractions with YG1021 S9 (B), YG1024 S9 (C), and YG1029 S9 (D). Subsequent analyses by GC–MS resulted in identification of 2-nitrofluoranthene, 1-nitropyrene, and 2-nitropyrene in subfraction 31, and benzo[a]pyrene and indeno[1,2,3-cd]pyrene in subfractions 21 and 24, respectively. Reproduced with permission from Legzdins et al. [18].

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anthene. Indirect-acting mutagens included triphenylene, benzo[a]anthracene, chrysene, benzo[e]pyrene, perylene, and benzo[a]pyrene. Schuetzle and Lewtas [1] generated mutation chromatograms of extracts of light-duty diesel particulates (SRM 1650) using NP-HPLC followed by GC–MS analysis to show that 30–40% of mutagenicity (TA98 S9) was attributable to seven nitro- and dinitro-PAH (1-nitropyrene, 3-nitrofluoranthene, 8-nitrofluoranthene, 1,3-dintropyrene, 1,6-dinitropyrene, 1,8dinitropyrene, and 2,7-dinitro-9-fluorenone. Thompson et al. [185] generated mutation chromatograms of the acidic fractions of extracts of air particulate prepared using a nonaqueous anion-exchange protocol. Air particulate samples were collected in Boise, Idaho, and contained significant amounts of material derived from wood combustion. The authors used a cyanopropyl HPLC column in concert with a NP gradient elution program to produce 1-min subfractions that were subsequently assayed using TA98 S9. Two distinct mutagenic retention time zones were identified; the first zone contained moderately polar compounds with retention times slightly longer than a 4-nitrophenol standard, while the second mutagenic zone contained polar compounds with retention times similar to a 2nitrobenzoic acid standard. DeMarini et al. [63,187] used the ion-exchange procedure of Thompson et al. [185] to fractionate municipal waste incinerator emissions; over 80% of the direct-acting mutagenic activity resided in the dichloromethane fraction. Mutation chromatograms were generated using an analytical scale NP-HPLC method using a cyanopropyl column; 1-min subfractions were assayed in TA98, TA98NR, and TA98DNP6. Based on the reduced responses of the TA98-active subfractions to TA98NR and TA98DNP6, the authors concluded that nitroarenes accounted for greater than 50% of the direct-acting mutagenic activity. In addition, one active subfraction exhibited an HPLC retention time similar to a 1-nitropyrene standard, implicating this compound as a primary mutagen in the extracts. DeMarini et al. [58] again used the TA98, TA98NR, and TA98DNP6 strains in a study of emissions from burning of scrap tires. Extracts were fractionated using an analytical-scale NPHPLC method (3 mm silica) and a pentane/dichloromethane/methanol gradient elution program; 1-min subfractions were collected and bioassayed to afford mutation chromatograms. The mutation chromatograms were similar in that four distinct zones of mutagenic activity were observed; subsequent analyses of the active composite subfractions resulted in identification of a variety of aromatic and mutli-ringed

mutagens, including PAHs and aza-aromatics. The authors concluded that dinitro-PAHs or aromatic amines accounted for the bulk of the direct-acting mutagenic activity, while PAHs accounted for much of the indirect (+S9) activity. Arey et al. [55] used mutation chromatograms in a bioassay-directed fractionation study of the mutagenicity of products of simulated gas-phase reactions of naphthalene, fluorene, and phenanthrene. Mutation chromatograms were generated by TA98 S9 bioassays of nine HPLC fractions from a semi-preparative silica column using a hexane/dichloromethane/acetonitrile gradient elution program. Using this approach, the authors identified nitro-PAH lactones as primary polar mutagens in the reaction product mixtures. Lee et al. [92] generated mutation chromatograms based on extracts of particulates from combustion of electric cables. Acetone extracts were fractionated on a Sephadex LH-20 column 15 mm i.d.  190 mm); individual 2-min fractions were assayed in TA98 S9. S9. The bulk of the mutagenic activity was confined to a narrow retention time range. Subsequent semi-preparative HPLC (10 mm i.d.  250 mm with 10 mm packing) of the active fractions followed by analytical scale RPHPLC (Nucleosil C18) afforded 1-min subfractions. The resulting mutation chromatogram exhibited two distinct peaks of activity, which were attributed to 1,6dintropyrene and 1,8-dinitropyrene using fluorescence spectrophotometry. Lewtas et al. [188] generated mutation chromatograms for mixtures of metabolites of 1-nitropyrene using a C18 RP-HPLC column (9.4 mm i.d.  250 mm) with a methanol/water gradient elution program. Thirtysecond subfractions were collected during 45-min RPHPLC runs using a fraction collector and subsequently bioassayed in two Salmonella strains. The aforementioned studies illustrate the effectiveness of mutation chromatograms in isolating and characterizing mutagens in extracts of complex environmental mixtures. Some key aspects of the methodology, as expressed in these studies, include the following: 1. Identification of specific compounds/compound classes in active subfractions through comparison of retention times of authentic standards, and ancillary analysis of active subfractions using mass spectrometric techniques, e.g., GC–MS. This approach has been particularly effective for the determination of nitroarenes as mutagens in air particulates. 2. Using mutagenic activities of authentic standards, and by determining concentrations of identified

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mutagens in active subfractions, the relative contribution to total mutagenic burden of a sample extract can be estimated. 3. Multiple levels of fractionation are frequently required to reduce the complexity of fractions prepared chromatographically to the point where individual mutagens or mutagen classes can be identified. Mutation chromatograms generated from fractions collected during narrow temporal windows (i.e., each minute) rapidly pinpoint the elution of mutagenic compounds, which greatly reduces the effort required to isolate and identify them in subsequent fractionation levels. 4. The use of different Salmonella typhimurium strains in generating mutation chromatograms can provide valuable insight into the chemical and biological characteristics of mutagens. 3.7. Other fractionation methodologies Additional fractionation methods have been developed and applied to airborne particulate samples. Kuba´tova´ et al. [156] used hot pressurized (liquid) water for fractionation of wood smoke and diesel exhaust particulate. Extraction and fractionation of analytes according to polarity was accomplished by sequential 30-min extractions at varying temperatures ranging from 25 to 300 8C; increasing temperature resulting in extraction of decreasingly polar compound classes. 4. Conclusions The coupling of short-term bioassays with analytical chemical techniques continues to be an effective tool for determination of mutagenic compounds in air particulate. It is apparent that the best methods not only effectively isolate mutagens, but also eliminate nonmutagens or ‘‘anti-mutagens’’ from the exceedingly complex extracts of air particulate material. Interestingly, many of the studies more recently reported in the literature continue to be based on classical methods developed 20 years ago, which speaks of the effectiveness and robustness of these tried and true fractionation techniques. Many of these open-column and HPLC methods were developed during the mid-1980s, during a significant shift in focus away from PAHs as the primary mutagens in air particulate, to more polar compounds including oxygenated compounds, nitro- and dintroPAHs, and other nitrogen-containing compounds. An excellent overview of mutagens identified in ambient air by compound class is reported in Claxton et al. [22]. The importance of these relatively polar compounds as

29

mutagens emphasizes the requirement for methods that effectively separate closely related compound classes, e.g., nitro- and dintro-PAHs, without significant crosscontamination between fractions. Although discussion of methods based on an initial acid/base/neutral partitioning scheme represents a significant component of this review, this procedure is now less commonly used, primarily as a result of relatively low recoveries of mass and mutagenic activity, and its reduced effectiveness for polar and/or acidic matrices. The cyanopropyl (or other chemically similar) stationary phase has appeared to supplant RP-HPLC and other NPHPLC phases for use in fractionating extracts of air particulate, which is due to its effectiveness and compatibility with the Ames assay. The Salmonella assay continues to be an effective tool for assessing mutagenicity of extracts of air particulates. Variations of this assay, e.g., microsuspension variant, continue to be developed in order to reduce the amount of material required to detect mutagenic activity. Any reduction in the material required for bioassay is important, given that small masses of extracted organic material available for chemical and biological characterizations are a chronic problem. More study of polar compounds associated with significant mutagenic activity is needed, as many of the studies reported in this review have demonstrated the importance of these compounds. The last few years have seen quantum leaps in development of technology amenable to analysis of polar compounds, most notably the coupling of liquid chromatography with mass spectrometry. The increased sensitivity of this new generation of LC/MS and LC/MS/MS instrumentation should also assist in overcoming limitations in sample quantities available for analyses. As of the writing of this review, application of these new LC/MS instruments to investigation of mutagens in air particulate have yet to be widely reported. Acknowledgements We thank Dr. Paul White for his invitation to contribute to this issue. Special thanks also go to Dr. Larry Claxton for his collegiality, encouragement, and provision of reference material. References [1] D. Schuetzle, J. Lewtas, Bioassay-directed chemical analysis in environmental research, Anal. Chem. 58 (1986) 1060A– 1075A.

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[2] L.D. Claxton, Review of fractionation and bioassay characterization techniques for the evaluation of organics associated with ambient air particles, in: R.R. Tice, D.L. Costa, K.M. Schaich (Eds.), Genotoxic Effects of Airborne Agents, Plenum Press, New York, NY, 1982, pp. 19–34. [3] L.D. Claxton, G. Douglas, D. Krewski, J. Lewtas, H. Matsushita, H. Rosenkranz, Overview, conclusions, and recommendations of the IPCS collaborative study on complex mixtures, Mutat. Res. 276 (1992) 61–80. [4] H. Matsushita, O. Endo, S. Goto, H. Shimizu, H. Matsumoto, K. Tamakawa, T. Endo, Y. Sakabe, H. Tokiwa, M. Ando, Collaborative study using the preincubation Salmonella typhimurium mutation assay for airborne particulate matter in Japan. A trial to minimize interlaboratory variation, Mutat. Res. 271 (1992) 1–12. [5] J. Lewtas, L.D. Claxton, H.S. Rosenkranz, D. Schuetzle, M. Shelby, H. Matsushita, F.E. Wurgler, F.K. Zimmermann, G. Lo¨froth, W.E. May, D. Krewski, T. Matsushima, Y. Ohnishi, G.C. Becking, Design and implementation of a collaborative study of the mutagenicity of complex mixtures in Salmonella typhimurium, Mut. Res. 276 (1992) 3–9. [6] W.E. May, B.A. Benner Jr., S.A. Wise, D. Schuetzle, J. Lewtas, Standard reference materials for chemical and biological studies of complex environmental samples, Mutat. Res. 276 (1992) 11–22. [7] H.S. Rosenkranz, W.T. Speck, Mutagenicity of metranidazole: activation by mammalian liver microsomes, Biochem. Biophys. Res. Commun. 66 (1975) 520–525. [8] M. Watanabe, M. Ishidate, T. Nohmi, A sensitive method for the detection of mutagenic nitroarenes: construction of nitroreductase overproducing derivatives of Salmonella typhimurium strains TA98 and TA100, Mutat. Res. 216 (1989) 211–220. [9] M. Watanabe, M. Ishidate, 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 (1990) 337–348. [10] M. Watanabe, P. Einisito, M. Ishidate, T. Nohmi, Mutagenicity of 30 chemicals in Salmonella typhimurium strains possessing different nitroreductase or O-acetyltransferase activities, Mutat. Res. 259 (1991) 95–102. [11] L.D. Claxton, M. Kohan, Bacterial mutagenesis and the evaluation of mobile source emissions, in: M.D. Waters, S.S. Sandhu, J. Lewtas-Huisingh, L.D. Claxton, S. Nesnow (Eds.), Short-term Bioassays in the Analysis of Complex Environmental Mixtures, vol. II, Plenum Press, New York, NY, 1981, pp. 299–317. [12] H.S. Rosenkrantz, E.C. McCoy, D.R. Sanders, M. Butler, D.K. Kiriazides, R. Mermelstein, Nitropyrenes: isolation, identification, and reduction of mutagenic impurities in carbon black and toners, Science 209 (1980) 1039–1043. [13] D. Schuetzle, T.E. Jensen, J.C. Ball, Polar polynuclear aromatic hydrocarbon derivatives in extracts of particulates: biological characterization and techniques for chemical analysis, Environ. Int. 11 (1985) 169–181. [14] S.A. Wise, C.F. Allen, S.N. Chesler, H.S. Hertz, L.R. Hilpert, W.E. May, R.E. Rebbert, C.R. Vogt, Characterization of air particulate material for polycyclic aromatic compounds, NBSIR 82-2595, National Bureau of Standards, Washington, DC, 1982. [15] S.A. Wise, S.N. Chesler, L.R. Hilpert, W.E. May, R.E. Rebbert, C.R. Vogt, M.G. Nishioka, A. Austin, J. Lewtas, Quantification

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