Environ. Sci. Technol. 1990, 2 4 , 890-894
S9-Activated Ames Assays of Diesel-Particle Extracts. Detecting I ndirect-Acting Mutagens in Samples That Are Direct-Acting James C. Ball, Barrle Greene, Willie C. Young, Joel F. 0. Rlchert, and Irving 1.Salmeen’
Research Staff, Ford Motor Company, Dearborn, Michigan 48121
We sought to detect S9-activated mutagens in dieselexhaust particle extracts by fractionating extracts using high-performance liquid chromatography (HPLC) and carrying out Ames assays (strains TA98 and TAlOO) with and without S9 mix on the eight fractions obtained thereby. S9 activation of the fraction containing unsubstituted and alkyl-substituted polycyclic aromatic hydrocarbons (PAH) was at the limits of detectability for strain TAlOO and undetectable for TA98. There was only one fraction that showed S9 activation; it was 20-fold more active with S9 mix. Six other fractions were strongly direct-acting and showed no enhancement of activity with S9 mix. The S9-activated mutagens detected when concentrated in a chromatographic fraction could not be detected in unfractionated extracts because the direct-acting mutagenicity of the other components dominated. We were unable to identify the indirect-acting mutagens from GC-MS data. Introduction This paper concerns a question about the use of the Ames assay to characterize mutagens in diesel-exhaust particle extracts; i.e., what can be said about “indirectacting” mutagens in diesel-particle extracts when the extracts are “direct-acting”. Here indirect-acting and direct-acting refer to assays carried out in the presence and in the absence of homogenized rat liver tissue, colloquially known as “S9 mix”. Many workers have shown that diesel-particle extracts are active in the absence of S9 mix ( I , 2), and that the addition of S9 mix changes the activity of the extracts only slightly (3-6). As Pederson and Siak (3) pointed out, Ames assays of these samples in the presence of 89 mix yield an unknown superposition of the activity pf those direct-acting mutagens unaffected by S9 enzymes, the activity of authentic promutagens activated by S9 mix, and the activity of direct-acting mutagens whose activity is modified by S9 mix. The distinction between indirect-actingand direct-acting mutagens is critical for using the Ames assay as a guide for research into the possible health effects of diesel-engine exhaust because it steers research toward two different classes of compounds. For example, Ames and co-workers found that various unsubstituted and alkyl-substituted polycyclic aromatic hydrocarbons (PAH), long associated with the carcinogenic activity of combustion products, were active only in the presence of S9 mix (7). Thus, when diesel-particle extracts turned out to be direct-acting in the Ames assay, there was naturally a turn of interest from the well-investigated PAH to a possible “new class” of carcinogens that the direct-acting mutagenicity suggested may be associated with combustion. Consequently, over the past 10 years or so, almost all of the efforts to characterize the mutagenicity of diesel-particle extracts have *Address correspondence to this author: Room S3083, Scientific Research Laboratories, Ford Motor Co., P.O. Box 2053, Dearborn, MI 48121. 890
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focused on direct-acting mutagens. The evidence now available indicates that nitroarenes account for most of this direct-acting activity (2, 8). Although the direct-acting mutagenicity of diesel-exhaust-particle extracts is now reasonably well understood, comparatively little is known about the indirect-acting mutagens in these extracts. To date, the cleanest demonstration that they contain indirect-acting mutagens detectable by the Ames assay has come from experiments with Salmonella strains resistant to mutation by nitroarenes (3). With these strains, the direct-acting activity of the extracts was attenuated sufficiently that activation by S9 mix could be detected. Here we report experiments with the standard Ames Salmonella strains TA98 and TAlOO in which we sought to characterize the indirectacting mutagens of diesel-particle extracts by chromatographically separating them from direct-acting ones. We separated the extracts into eight fractions by HPLC. For the classical PAH fraction, the S9-activated Ames assay activity was at the limits of detectability with TAlOO and undetectable with TA98, even though the fraction constituted -5% of the extract mass. A fraction that eluted between the classical PAH fraction and the nitropyrenecontaining fraction was weakly active without S9 mix, but showed activity 20X higher in the presence of S9 mix. This indirect activity, while easily detectable in the chromatographic fraction, was too weak to be detectable in unfractionated extracts because the direct-acting activity of the other components dominates. The other six fractions were direct-acting, and their activity was not enhanced by S9 mix. Materials and Methods Diesel-exhaust particles were collected on filters in a dilution tunnel with a vehicle run on a chassis dynamometer through 4 cycles of the 1974 Federal Test Procedure hot cycle. The vehicle was a 1984 Escort with a 2.0-L direct-injection diesel engine. The filters were extracted in a Soxhlet apparatus with dichloromethane in a nitrogen atmosphere. The extracts were separated into eight fractions by normal-phase high-performance liquid chromatography (HPLC) (Varian Vista 5500 system; Whatman Magnum 9 normal-phase silica gel 50-cm column) using a hexane to acetonitrile gradient elution sequence. All of these procedures were the same as those described in ref 2.
Ames assays with and without S9 mix were carried out following the procedure of Maron and Ames (9). Rat liver S9 homogenate, prepared from Arochlor-1254-inducedrats, was purchased from Microbiological Associates, Inc. (Rockville, MD). Four concentrations of S9 mix were used. These were prepared by adding 1, 2, 5, or 20 mL of S9 homogenate to the mixture buffer, salts, glucose 6-phosphate, and NADP such that the final volume was 50 mL. The corresponding S9 concentrations were 2, 4, 10, and 20%. The “standard” S9 concentration is 4%. Ames assays were carried out by adding 500 p L of S9 mix to the plates. The activity of the commercial S9 homogenate was tested by using 4% S9 mix and benzo[a]pyrene (BaP) for
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MICROGRAMS EXTRACT PER PLATE Figure 1. Ames assay data with strain TA98 for chromatographic fractions of diesel-particle extracts. The sample concentrations, lndlcated as micrograms of extractlplate, correspond to the mass of each fraction. Data corresponding to open and closed circles were obtalned in the presence of and in the absence of S9 mix, respectively, Le.,indlrect-actlng and direct-acting. Data indldated by crosses for the unfractlonated extract (upper left panel) correspond to the presence of S9 mix, but wRh the NADP cofactor omitted. Chromatographic fractions, indicated by the numbers on the individual panels, are discussed In the text. Data for fraction 1 are not shown because the response was not dlfferent from the spontaneous revertant level. Data shown are average of duplicates. The difference between the two polnts was typically less than 5 % of the average. Lines between the data points are to help visualize the data, and have no theoretical meanlng.
TAlOO and 2-aminofluorene (2AF) for TA98. Typical results for these standards were strain TAlOO BaP (rdplate) 0 0.25 0.50 1.0 2.0 revertant/plate (duplicates) 110 322 422 574 538 105 312 446 600 603 strain TA98 2AF ( d p l a t e ) 0 revertant/plate (duplicates) 63 45
3.0 602 594
0.20 0.50 1.0 2.0 112 297 529 1289 107 283 486 1142
The results for these standards were essentially the same as those reported by Maron and Ames (9),which affirmed the activity of the S9 mix. Every experiment using S9 mix included dose-response data for these positive controls with duplicates at each compound concentration. Positive controls for experiments without S9 mix were methyl methanesulfonate (TA100) and 2-nitrofluorene (TA98). Photomicrographs of the background lawn were taken to assess cell killing (10). Gas chromatography-mass spectrometry (GC-MS) data were obtained with a Hewlett-Packard Model 5970 GCMS system. The samples were injected onto an HP-1 fused-silica capillary column (12 m X 0.2 mm X 0.33 Mm film) in the splitless mode. The GC oven was temperature programmed for an initial temperature of 40 "C (2-min hold), followed by ramping of the temperature at 4 "C/min to 300 "C, followed by holding at 300 "C for 20 min. Mass spectra were obtained in electron impact mode a t 70 eV and source temperature of 200 "C. The mass range scanned was 45-500 amu. Concentrations of selected PAH were estimated by comparing integrated GC-MS peaks for the chromatographic fractions of the diesel-particle extract
samples with the integrated GC-MS peaks recorded for a mixture of reference PAH.
Results Ames assays of unfractionated extracts were carried out first (Figures 1and 2,upper left panels). The direct-acting activities of the unfractionated diesel-particle extracts were comparable to those reported previously (2) for samples prepared in a similar manner. The indirect-acting activities of unfractionated extracts were similar to those reported by others (1, 3-6). Specifically, mutagenicities obtained with S9 mix (4%) were slightly lower than those obtained without S9 mix. The response obtained for TA98 by omitting the NADP cofactor from the S9 mix was about the same as that obtained in the presence of the cofactor (Figure 1, upper left panel), indicating that the slight decrease in mutagenicity in the presence of S9 mix was not due to NADP-dependent enzyme activity. For a fixed amount of unfractionated extract (80 pg/plate), the Ames assay activity (TA98, data not shown) decreased from 800 revertants/plate with no S9 mix to -400 revertants/plate with 4% S9 mix, and remained at -400 revertants/plate with 10 and 20% S9 mix, consistent with observations of Pederson and Siak (3). Thus, by the criteria of Ames assays, the unfractionated extracts for the present study were similar to those studied earlier by us and by other investigators. Fraction 1comprised -60% of the extract mass. It was not mutagenic and consisted of paraffinic compounds derived possibly from lubricating oil or from unburned fuel. Fraction 2 accounted for -5% of the extract mass. For strain TA98 this fraction was mutagenic (lo0 revertants/pg) of fractions 4 and 5 were due to these nitroarenes. Fraction 8 accounted for -20% of the extract mass. For strain TA98, the mutagenic activity in the presence of S9 mix was about half of that observed in the absence of S9 mix. This reduction was not investigated. For strain TA100, fraction 8 at concentrations 400 and 800 pg/plate was strongly cytotoxic in the absence of S9 mix, as evidenced by the strongly nonlinear concentration-response curve, and by a very sparse background lawn. The S9 mix reduced this cytotoxicity. The different cytotoxic response elicited from the two strains, while interesting, was not investigated. Very little is known about the compounds in fraction 8, except that they are polar, as indicated by the solvent (acetonitrile) needed to elute them from the column. The compounds in fraction 8 do not separate well by standard normal or reverse-phase chromatography on silica, and most of them do not elute from GC columns (2, 11, 12). Identification of compounds in this fraction remains as an unsolved analytical chemistry problem. The mass recovery following the chromatography was 93 f 5 % , averaged over five different separations. These mass recoveries were similar to those we achieved previously (2). The complete fractionation procedure and Ames assays were carried out twice, using the same extract. The results were the same.
Discussion These experiments lead to three conclusions. (1)The concentrations of classical promutagenic PAH, that is, unsubstituted and alkyl-substituted PAH, were near or below the detection limit of the conventional Ames assay. (2) The samples did contain promutagens that were strongly activated by S9 mix (fraction 3). We were unable to identify these promutagens, although GC-MS data showed that classical PAH were not detectable in this fraction. (3) The indirect-acting mutagenicity of these diesel-particle extracts was too low with respect to the “direct-acting” activity to be detectable with “standardn Ames assays of the unfractionated extracts. The first conclusion is consistent with our measurements of PAH concentrations in fraction 2. For example, the concentration of BaP was around 1.2 f 0.5 ng/pg of fraction 2 (Figure 3). For strain TA100,lOO pg of fraction 2, yielded -150 k 50 revertants above the spontaneous revertant level; the BaP in 100 pg of fraction 2 could account for -50 f 25 revertants (see data for BaP under Materials and Methods). The S9-activated mutagenicity of chrysene in TAlOO is -38 revertantslnm (7). The concentration of chrysene in fraction 2 could not be accurately determined because it coeluted with triphenylene. The concentration of chrysene and triphenylene together was 6 f 2 ng/pg of fraction 2. Other investigators have reported that the concentration of chrysene in vehicle exhaust samples is typically around twice that of tri-
phenylene (11). If we assume that in our samples the ratio of chrysene to triphenylene was -2, the chrysene concentration would be around 4 i 1ng/pg of fraction 2, and the chrysene in 100 pg of fraction 2 could account for 60 f 20 revertants. Triphenylene is not mutagenic. The Ames assay activities of other unsubstituted and alkylsubstituted PAH (e.g., dibenzanthracene, benzanthracene, 7-methylbenzanthracene, 7,12-dimethylbenzanthracene) are 5-10 times less than that of BaP (7), and the concentrations in diesel-particle extracts of these other PAH are generally considerably less than that of BaP or chrysene (11). Thus, the other known mutagenic PAH could account for in the order of a few tens of revertants. The reliability of this accounting, however, is unknown since the effects of the complex mixture on the activation by the S9-mix enzymes are unknown. The second conclusion directs attention to promutagens other than classical PAH. The promutagens in fraction 3 have not been identified. The prominence of 9-nitroanthracene and methylnitroanthracene/phenanthrenein the GC-MS data for this fraction suggests, as a working hypothesis, compounds of this type as candidates, although 9-nitroanthracene is only weakly mutagenic with S9-activation (13). On the other hand, some akylnitroaromatics of a similar type are weakly direct-acting, but are modestly active in the presence of S9 mix (14). The third conclusion follows from the data for fraction 3, the only fraction that showed mutagenicity activated by S9 mix. For example, the maximum number of TAl00 revertants obtained for fraction 3 was 600 (at 20 pg/plate), and this fraction accounted for 1% of the total extract mass. The direct-acting mutagenicity for 100 pg of the unfractionated extract was -1000 f 50 revertants. If we assume that the direct-acting mutagens were unaffected by the S9 mix, then in the presence of S9 mix, 100 pg of the extract would yield lo00 f 50 TAlOO revertants from direct-acting compounds and -30 revertants from the indirect-acting compounds in fraction 3. This small difference is not detectable within the fluctuations of the Ames assay. A similar conclusion obtains for strain TA98. Thus, the indirect-acting mutagens would be undetectable in assays of the unfractionated extracts. The final point for discussion is to call attention to recent experiments by Grimmer et al. (15) in which material obtained from diesel-engineexhaust was fractionated and tested for carcinogenicity in a rodent lung implantation assay. These investigators found that essentially all of the carcinogenicity of their samples could be attributed to four- to seven-ring PAH. Since many carcinogenic PAH are mutagens in the S9-activated Ames assay, we are led to wonder if the barely detectable S9-activated mutagenicity of our fraction 2, Le., the PAH fraction, suggests an inconsistency with the data of Grimmer et al. A rigorous answer is not possible with the data at hand because we and Grimmer et al. studied different types of samples, and we used different fractionation schemes. Our samples were organic solvent extracts of particles collected on filters in a diluted exhaust stream; those of Grimmer et al. were from the whole condensate of undiluted exhaust, including washings of the heat exchanger and extracts of the combustion-produced water. We fractionated samples by HPLC; Grimmer et al. fractionated their samples by a solvent-solvent extraction, followed by Sephadex LH-20 and silica gel chromatography. Our fraction 2 contained many of the same four- to seven-ring PAH compounds isolated by Grimmer et al. and, to within a factor of 2, in similar concentrations. We have no way of knowing, however, whether or not the S9-activated mutagens were
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Environ. Sci. Technol. 1990, 2 4 , 894-897
isolated in our fraction 3 would have occurred in the fraction in which Grimmer et al. isolated four- to seven-ring PAH compounds. We also cannot rule out the possibility that the Ames assay fails to detect the carcinogens that were detected in the bioassay used by Grimmer et al.
(8) Ball, J. C.; Young, W. C.; Salmeen, I. T. Mutat. Res. 1987, 192. 283-287. (9) Maron, D. M.; Ames, B. N. Mutat. Res. 1983,113,173-215. (lo)S h e e n , I. T.; Durisin, A. M. Mutat. Res. 1981,85,109-118. (11) IARC International Agency for Research on Cancer.
Literature Cited Claxton, L. D. Environ. Mutagen. 1983, 5, 609-631. Salmeen, I. T.; Pero, A. M.; Zator, R.; Schuetzle, D.; Riley, T. L. Environ. Sci. Technol. 1984, 18, 375-382. Pederson, T. C.; Siak, J.4. J. Appl. Tonicol. 1981, I , 61-66. Lofroth, G. Environ. Int. 1981,5, 255-261. Lewtas, J. Mutagenic Activity of Diesel Emissions; Lewtas, J., Ed.; Elsevier Science Publishing, Inc: Amsterdam, 1982;
46. (12) Bayona, J. M.; Markides, K. E.; Lee, M. L. Enuiron. Sci. Technol. 1988,22, 1440-1447. (13) Fu, P. P.; VonTungeln, L. S.; Chou, M. W. Carcinogenesis 1985,6, 753-757. (14) Rosenkranz, H. S.; Mermelstein, R. Mutat. Res. 1983,114, 217-267. (15) Grimmer, G.; Brune, H.; Deutsch-Wenzel, R.; Dettbarn, G.;
pp 243-264.
Cooper, B. J.; Shore, P. R. Society of Automotive Engineers, 1989, PaDer No. 890494. McCk,-J.; Choi, E.; Yamasaki, E.; Ames, B. N. h o c . Natl. Acad. Sci. U.S.A. 1975, 72, 5135-5139.
Evaluations of Carcinogenic Risk to Humans. Diesel and Gasoline Engine Exhaust; IARC: Lyon, France, 1989; Vol.
Jacob, J.; Naujack, K. W.; Mohr, U.; Ernst, H. Cancer Lett. 1987, 37, 173-180.
Received for review October 17, 1989. Revised manuscript received January 26, 1990. Accepted February 15, 1990.
Atmospheric Chemistry of Gaseous Diethyl Sulfate Steven M. Japar, Timothy J. Wallington, Jean M. Andino, and James C. Ball
Research Staff, Ford Motor Company, Dearborn, Michigan 48121 The atmospheric reactivity of diethyl sulfate (DES) has been investigated. Upper limits to the rate constants (in cm3 molecule-' 5-l) for the homogeneous gas-phase reactions of DES with 03,NH3, and H20 have been determined by FTIR spectroscopy and are C3.4 X C1.4 X and 12.3 X respectively. The reactivity of DES toward OH radicals and C1 atoms has been determined by using relative rate techniques; rate constants for those reactions are (1.8 f 0.7) X and (1.1f 0.1) X lO-l', respectively. These rate constants correspond to atmospheric lifetimes ranging from 11 day with respect to reaction with water to >12 years with respect to ozone. With the possible exception of its reaction with water, these results indicate that the atmospheric fate of DES within an urban air parcel is not determined by its homogeneous gas-phase reactions with any of the atmospheric species studied. No evidence has been found for the formation of DES or related compounds during the ozonolysis of olefins in the presence of SO2 and ethanol. Introduction Diethyl sulfate (DES) has been shown to be carcinogenic in rats ( 1 , Z ) and is mutagenic in Salmonella typhimurium strain TAlOO (3). Because it is one of the most potent carcinogens of the class of simple alkylating sulfuric acid esters, it has been studied in order to investigate the relationship between chemical reactivity and carcinogenicity (2). Although DES has not been identified in the atmosphere, its methyl analogue, dimethyl sulfate (DMS), has been detected in the gas phase at ppb levels in Los Angeles air ( 4 ) and in power plant plumes (5,6). Since it has been speculated that the formation of DMS in the atmosphere involves the reaction of methanol with sulfuric acid aerosol (4-61, a concern has developed that centers on the possibility that atmospheric methanol levels may rise significantly with increasing use of methanol and methanolblended fuels for motor vehicles. Since ethanol and ethanol-blended fuels are used extensively in a number of areas, notably Brazil, and since their usage may increase 894
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significantly in the United States in the future, a similar concern about atmospheric levels of DES may develop. We have recently completed a detailed study of the formation and reactivity of DMS in the atmosphere (7,8) in which it was shown (1)that DMS was not formed via homogeneous oxidation reactions involving methanol and SO2, (2) that it could be formed through a probable heterogeneous reaction of SO3with dimethyl ether, and (3) that its homogeneous gas-phase reactions with OH, C1, 03, and NH, were too slow to control its atmospheric fate. This report details a similar study of the atmospheric chemistry of DES. Experimental Procedures Experiments were carried out in a system consisting of a Mattson Instruments Inc. Sirius 100 FT-IR spectrometer interfaced to a 140-L, 2-m-long evacuable Pyrex chamber described previously (7-9). The Pyrex chamber was equipped with White-type multiple-reflection optics (path length set up to 34.2 m), and was surrounded by 22 UV fluorescent lamps (GTE F40BLB). The spectrometer was operated at a resolution of 0.25 cm-'. Infrared spectra were typically derived from 8 to 16 coadded interferograms. Reference spectra were acquired by filling the chamber with known concentrations of the appropriate compounds. We have used the relative rate technique to investigate the kinetics of the reaction of DES with C1 atoms and OH radicals in the evacuable chamber (7,8). C1 atoms were generated from the photolysis of C12 by the black lights, while OH was formed through the photolysis of methyl nitrite (CH30NO)/N0 mixtures in air (10). Reaction mixtures consisting of a reference organic, DES, and C1, or CH,ONO/NO were introduced into the chamber by expansion of the gases from known volumes, diluted with synthetic air, and left to mix for at least 5 min. In the presence of C1 atoms (or OH radicals) there is competition between DES and the reference organic for the available reactive radicals via reactions 1 and 2, i.e., for C1 atoms C1 + DES products (1) C1 + reference organic products (2)
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