Ames Testing for Mutagens and Carcinogens in Air Peter Flessel, Yi Y. Wang, Kuo-In Chang, and Jerome J. Wesolowski Air and Industrial Hygiene Laboratory, California Department of Health Services. Berkeley, CA 94704 Human exposure to toxic chemicals is a fact of life. The challenee is to determine the most serious risks amone the multituhe of exposures. In the State of California's ~ i b l i c Health Laboratory, we have focused on public health risks associated with involuntary exposures to chemical mutagens and carcinogens in air. This paper will describe a laboratory measurement approach that integrates the Ames mutagenicitv test ( 1 ) with chemical analysis for evaluating- mutagenic and carcinogenic chemicals in air. Deflnlng the Problem Chronic exposure to low levels of mutagens and carcinogens in the air occurs because of everyday releases from such sources as vehicles, incineration processes, chemical manufacture. consumer oroducts. and buildine materials. What is the magnitude of the air cancer risk and which pollutants and sources nose the ereatest risks? Using available exnosure data and risk assessment models, EPA has provided some preliminary answers (2). Ambient air exposures are associated with between 1,000 and 2,000 cancer cases per year nationwide, a small fraction of the more than 800,000 total cases per year. Most cancer cases are attributed to exposures through diet and smoking, according to the estimates of Doll and Peto (3).These rough estimates also suggest that about 2% of total cancers (about 4,000 cases annually) are associated with involuntary exposures to chemical pollutants in air and water (3).Thus carcinogens in air may contribute UD to one-half of the cancer cases attributable to carcinogens in air and water. (Since nearly all cancer cases have multinle causes. the above attributions are onlv statistical.) The highest risk pollutants are found in the particulate products of incomplete combustion (2). These include the polycyclic aromatic hydrocarbons (PAH) and their nitrated derivatives, the nitroarenes (N02PAH). Emissions from road vehicles and residential heating contribute over half of the air cancer risk in the United States while industrial emissions contribute between 20 and 25% (2). This source apportionment of cancer risk a t the national level is consistent with recent mutagen source apportionment for Newark,
Presented at the Annual Meeting of the American Chemical Society, Anaheim. CA, September 8, 1986.
NJ, by Daisey and co-workers (4). In Newark, emissions from vehicles contributed about half of the direct-acting mutagenic activity of the nonpolar particulate organic matter. Measurement Uncertainties .Air cancer riskassessments aresubjert tomany uncertainties. l'herr are two maior kinds: those related to the assessment of the risks and those related to the measurements of carcinogens. Current risk assessments rely heavily on extrapolations of animal cancer potency data to humans. They estimate lifetime human exposures primarily from pollutant emissions data and disnersion modeling. Such risk assessments tend to be "conservative", i.e., they tend to overestimate the maenitude of the risk in order to he nrotective of the public hialth. On the other hand, the measurement uncertainties nrohahly underestimate the magnitude of the problem, mainly because it is not technicauy feasible to measure all the carcinogens in emissions or in the air. We will discuss only 1ahorator;measurement problems here. The chemical complexity of air emissions is the first and foremost challenge to our measurement strategy. Outdoor air samples contain dozens, perhaps even hundreds, of toxic chemicals in trace amounts. I t is clearlv not feasible to measure each one of these chemicals directly by chemical means. This can be a problem for risk assessment since some chemicals may be present in very small amounts but may be very potent carcinogens. These chemicals will not be picked up by chemical analysis unless there are specific reasons to look for them. Furthermore, measurement uncertainties related to the complexity of air apply both to compounds in the particulate and vapor phases. Thus a t the present time, risk assessors are attempting to evaluate the cancer hazards of air when most of the carcinogenic pollutants are not measured! A second major source of measurement uncertainty relates to atmospheric transformations. Chemical reactions of air nollutants mav increase (or decrease) the carcinoeenic of air. For example,'formaldehyde, an animaicarcinogen, is produced in significant amounts by atmospheric reactions (2). EPA's current cancer risk assessment does not take into account atmosoheric transformations because it is based largely on emissions data. Another potential source of uncertaintv concerns interactions of chemical carcinogens in air. ~ " r r e n tgovernment strategies assume that the risks are additive (2). However, it
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is known that exposure combinations can sometimes be more (or less) riskv than the simnle sum of the individual risks. A well-known case of synergy involves smoking and exposure to asbestos. Both predispose people to lung cancer, hut individuals who both smoke and are exposed to asbestos have a lung cancer risk that is approximately the product (not the sum) of the two individual risks. A fourth source of uncertainty in the current assessment of air cancer risk is the failure to consider indoor air exposures (2). This is a serious limitation in describing the total air cancer picture because time studies show that most people spend more than 90% of their time indoors. Further, the indoor concentrations of manv- oollutants (e.e.. . . " tobacco smoke, formaldehyde, many volatile solvents, radon) are greater than the outdoor concentrations. (Although indoor air pollution is a critical problem, it is beyond the scope of the present discussion, which deals with outdoor air pollution. See reference 5 for a summary of efforts by California's Health Department in this area.)
The Ames Test as a Prescreen for Cancer-Causlng Chemicals
In evaluating air samples, we would like to know which ones contain the highest levels of cancer-causing chemical (6).Animal bioassavs are too exoensive and time-consumine for routine screenkg, while chemical testing alone is not feasible because of the complexity of air. We have therefore used the Ames test to investigatethe mutagenic potential of air because many carcinogens are also mutagenic. The Salmonellalmammalian microsome test ( 1 ) developed by Bruce Ames and his colleagues a t the University of California, Berkelev. measures the abilitv of chemicals to nroduce mutations sensitive strains ofbacteria. The re& with the test strongly support the somatic-cell mutation theory of cancer. In a number of validation studies involving several hundred chemicals. nearlv four out of everv five animal carcinogpns assayed wrre found to he mutagenic in the Ames test (71.This is the reason whv the test has utility in cancer risk &aulation. Although th; test's sensitivity depends to some extent on the chemical class (ex., halogenated oreanic carcinogens are known to give false-negat&es), there is a good correlation for carcinogenic PAH and nitroarenes present in particulate organic matter (POM). Of particular importance for public health, there are no false negatives (i.e.. . . carcinoeens that are not mutaeenic in the Ames test) among PAH and NOpAH found in communiry air. In oractice. the test s a t n ~ l e a n dabout one hundred million bacteria unable to synthesize histidine (His-) are added to a test tube containine soft aear. Often a mammalian liver extract is added to mimic &man metabolism and thus to detect mutaeenic activity of chemicals requirine metabolic i conactivation. The mixtureis transferred to ~ e t i dish taining hard agar, salts, and glucose and incubated for several days. If the sample contains mutagens, some of the histidine-requiring cells are reverted to the (His+) wild type, histidine biosynthesis resumes, and the growth of visible revertant colonies is seen. An increase in the number of colonies (over the number of spontaneous background revertants) indicates that the chemical is mutagenic while the number of revertant colonies orovides an index of the mutagenic power of the sample. 1; air pollution studies, the test results indicate that mutagens are readily detectable in urhsn air. A geoxraphic comparison of mutagenic activity in air samples collerted at three California locations is shown in 1. Clearly, many more revertant colonies are seen in ~ i ~ & e the aerosol extract from Bakersfield, near major oil fields, than in the extracts from Sacramento, a nonindustrial city. No increase in revertants (over the filter blank) is seen a t Lakeport, a rural location.
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The Ames Test in the Study of Airborne Mutagens and Carcinogens
Our hypothesis is that the Ames test has many useful applications in the evaluation of airborne mutagens and carcinogens. To evaluate the hypothesis, we began by describing the problem of airborne carcinogens. Now we hope to show how the Ames test can complement chemical analysis in addressing the problem. Clarification of Measurement Uncertainties
Are current air uollution control strateeies controlline carcinogenic pollutants? The Ames test proGides a rough s k o eate for the total carcinogenic uotential of an air samde because it measures the iota1 mutagenicity of the sample. Thus, it provides a gross index against which specific chemical concentrations can be compared. I t can tell us whether the total amounts of mutagenic and potentially carcinogenic chemicals are remaining constant over time or are increasing as the result of increases in the concentrations of very potent carcinoeens that mav be Dresent in verv low amounts. In this way, t h e ~ m e test s can he useful in detkrmining whether the fine-tuning of the current control strategy, designed mostly to minimize photochemical smog production and visibility degradation, also decreases or perhaps even increases levels of chemical mutagens and carcinogens. The Ames test can also be used as a screening tool to provide information on atmospheric transformatio&. As described above, much research is needed to evaluate the significance of atmospheric transformations for purpose of cancer risk assessment. Clearly, definitive mechanisms can he elucidated onlv throueh detailed chemical analvsis. However, Ames testing can reveal the magnitude and direction of chances in the mutaeenic uotential of air samoles. Currentlv the test is being applied in this way to study atmospheric transformations of mutagenic nitroarenes by Pitts and coworkers (8) and wood smoke mutagens by Kamens and coworkers (9). Finally, the Ames test can help resolve problems of synergy and antagonism that compound-by-compound chemical
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Figure 1. Geographic comparison of mutagenic activity in air particulate mane,. Twentytour-hour high-volume samples were collected on glass-tiber filtersat sites in Bakersfield. ~~~~~. Sacramento.and Lakeoon. California.on Novem~~~~~
be, 2. 1976. F ltern were extracted wilh acetone an0 lne organlcexlractaole materm assayed for rnuagsn c act w t y in Salmonella TA98 wth melaoo ic act vat on ( A r m or 1254-maxed rat lher SO us descrioea n reference 10 Each plate received the extract from approximately 75 m3 of air.
analvsis alone cannot. Because the Ames test detects the mutagenicity of the total sample, it is possible systematically to investigate interactions between eroups - - of chemicals in complex a-ir mixtures. Ames Testing of Air Samples by the California State Health Department Laboratory Many university and government laboratories use the Ames bioassay as an instrument for conducting research on and environmental oollution orohlems involvine mutaeens " carcinogens. The studies by our laboratory reviewed below emphasize public health aoplications of the test to commu.. n i t i outdoor air. Geographic Distribution of Aerosol Mutagens in a n Industrial County: Integration of Ames Test Results in an Epidemiological Cancer Study. We have measured the geographic distribution of aerosol mutagens in outdoor community in an industrial northern California county and integrated the Ames test results with chemical analysis in an epidemiological cancer study (10). The application of the Ames test to e~idemioloeicalcancer studies is loeical hecause mutagenicity is in some measure a composite index of total potential carcinogenicity. The cancer study was carried out in collaboration with the California Department of Health Services Resource for Cancer Eoidemioloev and the EPA (11). Its purpose was to examine the relat%nship of lung cancer incidence to ambient levels of air pollution in Contra Costa County, California. It was hypothesized that the presence of heavy industry, mainly petrochemical plants and oil refineries, could he a contributing factor. Air particulate samples were collected a t 15 stations throuahout the countv for one "vear.. and orwnic extracts of the particulate matte; (POM) were prepared and analyzed for mutaeenicitv and selected PAH. Details of samoline . and analysis &ay be found in reference 10. Other pollutants including lead (Pb) and sulfate (SOa2-) were also measured. In order to compare the cancer incidence data to the air
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pollntion measurements, information atwut geographic variair pollution in the countv was ations in levels of community~. needed. Computer maps showing the geographical disirihution of the levels of the measured pollutants were constructed using SYMAP, a program in which the sampling station coordinates and associated pollutant levels were the input variables. These eeoeraohic " - . distributions were used to estimate community exposures, which in turn were compared with the distribution of cancer derived from e~idemioloeical studies. ~
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Figure 2 shows contour maps for Pb, Sod2-, coronene, and mutagenicity (TA98+S9) measured in composite samples collected during the winter months (November-February) 1978. Note the similarity of the geographical distributions of Pb, derived largely from auto emissions, and coronene. The patterns for several other PAH (not shown) were similar to that of coronene. Comparison of the contours indicates that vehicular emissions are undoubtedly significant contrihutors to PAH. In contrast to Pb, the 5 0 4 % - distribution is different. I t runs rouahlv in an eastiwest direction alone the industrial corridor inthe northern part of the county. i cis is consistent with the fact that SO,, the precursor of SOL2-. . . is emitted mostly hy stntionarysou;ces (inrludingpetrorhemical refincries and power plants) located alonc the industrial belt. The mutagenicity pattern ismore compikx, hut it shows a greater similarity to the Ph pattern than to the SOa%pattern. The similarity between the P b and mutagenicity contours suggest that mobile sources are contributors to particulate mutagens, as well as PAH. The correlation analysis of lung cancer rated by census tract and various air pollution constituents showed only one statistically significant relationship. That relationship was between Sod?- and lung cancer in males, hut not in females. However, when controlled for the percentage of the population categorized as blue collar workers the relationship was eliminated. I t was concluded, therefore, that there was no detectable effect on lung cancer risk from anv measured constituent of air pollution. While this study did not esCONTRA COSTA. NOV.78-FEB.79 tablish community air links to lung cancer, i t did demonstrate the feasibility of using the Ames test for determining the geographic distributions of particulate mutagens in ambient air and integrating this information in an epidemiological cancer study. We also note that the mutagen and PAH levels measured in Contra Costa County air were consistent with the negative epidemiological findings. From the measured levels we calculated the daily doses due to mutagens and PAH in airborne particles. Then we compared t h e doses t o t h e amounts in cigarette smoke particles. Breathing Contra Costa air gave a daily dose about equal to the dose from one-tenth of a cigarette (12). Lung cancer effects from smoking a tenth of a cigarette per day would not have been detected by the epidemioloei. cal study. Figure 2. Pollutant contour maps. Air particulate samples were collected at 15 statlons in Contra Costa County. California,and analyzed for mutagenic and chemical pollutants. Pollutant levels and sampling station cwrdlnstes Finally the study pointed were used to construct maps with a computer program called SYMAP (to). up the advantage of combin-
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ing the Ames test and chemical analysis. In addition to coronene, we measured four other major PAH; benzo(a)pyrene (BAP), benz(a)anthracene, henzo(ghi)perylene, and chrvsene. When we compared the contribution of the measurkd PAH to the mutagenic activity observed, we found that the sum of activities of these PAH represented only about 2% of the total mutagenicity of the air particulate matter. Thus much more complete chemical characterization of particles in ambient air was required to order to account for the mutagenicity as measured by the Ames test. Sources of Mutagens in Communitv Aerosols: Diurnal Variations and elations to Source missions Tracers. Subsequent studies, supported in part by the California Air ~ e s o u i c e sBoard, were-carried out to define the sources of particulate mutagens better (13). These studies used the Ames test to examine the relations between mutagenic aerosols and other air pollution variables, including certain source tracer elements. Ambient aerosols were sampled during pollution episodes in Contra Costa Countv between 1981 and 1081. Mutaeenic activity of aerosol e x t r k t s measured in the Ames test was compared with PAH content and elemental source tracers for vehicles (Pb), industry (Ni), and soil (Fe). Mutagenicity was found to be consistentlv and strnnelv associated with lead-containing fine (less than i.5 r m aerodynamic diameter). Thus vehicular transportation sources were predominant contributors to ambient air particulate mutagens during these pollution episodes in Contra Costa County.
Martinez. California
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We also examined the diurnal variation of mutagenirity in fine nirborue particles (14). The results of one field study conducted in Martinez. California. at a location adiarent to hoth stationary and mobile sources is shown in Figure 3. Hieh-resolution diurnal measurements of mutaeenicitv were done using a modified Ames test, the micropreincub;tion procedure of Kadoet al. (15). Thismodified Ames test is approximately an order of magnitude more sensitive than the standard plate incorporation test so can he applied when sample amounts are limiting. The diurnal pattern exhibits hoth early morning and late evening peaks that are characteristic of the daily traffic pattern. Stationary source emissions of SOzfrom the nearby refinery did not exhibit diurnal changes. ~urthermore,lead and mutagenicity were strongly correlated (r = 0.92), as shown by the plot of samplevalues in Fieure 4. We concluded that mobile source emissions cont r s u t e d significantly to the mutagenicity of airborne particles sampled in Martinez during this episode. The conclusion that vehicles are major mutagenic sources is consistent with those drawn by Pitts and co-workers (16) in southern California and Daisey co-workers (4) in the New York metropolitan area. Finally we found no evidence that refineries contributed significantly to aerosol mutagens. Nickel is a tracer for fuel ofi comhus&on and r e f i n e r ~ b ~ e r a t i o No n . statistical relationship was found between nickel and aerosol mutagenicity. Our failure to establish clear links between stationary source emissions and particulate mutagens is in contrast to findines of Gibson (17). . . His work indicated that stationarv industrial emissions are major mutagenic sources in Detroit. Seasonal Variations and Trends in Concentrations of Air Particle Mutagens. The results of long-term monitoring studies provide critical baseline information against which the impact of new or expanding technologies (e.g., diesel cars, solid-waste incineration) can he measured. From a pub-
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Fioure 3.Diurnal variation of mutaoeniciw of fine airborne oarticles. Tw~hour sampler were co leclea n Mart nez, Californna,and measJred in T A 9 8 with S9 usmga mod fleaAmes test procodure(redrawnfromref 14. w thpermisslonl. ~
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Figure 5. Trends and seasonal variations in mutegenicliy and benzo(a)pyrene concentrations.Test samples were prepared from hi-vol f i k s collected every sixth day at three sampling stations in Contra Costa County (Richmond, Concord. Pimburahl. Seoarate com~ositesfor each station were DreDared an0 ana yzed for mLtagen,c acllvlly tn TA98ano BAPano the resu,tsaversgw to o m n m e seasonal compar8te va ues shown
lir health perspective, such monitoring is essential to identify trends in the levels of toxir air contaminants. To obtain informationahout thc seasonal variar~onsandtrend*, Hi-rol air samples were collected every sixth day at thrw sampling stations iltichmond. Concord. Pittsbure) in Contra Cnsta County. Four-month composite samples were prepared to correspond with the three meteorological seasons in the Bay Area (July-October; Novemher-February; March-June). Air filters were extracted and the extracts tested for mutagenic activity in TA98 and analyzed for PAH by high pressure liauid chromatoara~hvas described in reference 10. seasonal cornposit;? r<s for the two-year period from July 1984 to June 1986 are shown in Figure 5. Seasonal variations in direct-acting mutagenicity (that observed in the absence of metabolic activation) are compared with levels of BAP measured in ambient aerosols. Dramatic seasonal variations in the levels of both mutagenicity and BAP were observed. Concentrations of both pollutants were highest in winter. Studies in New Jersey also have shown that BAP levels and mutagenicity of air particulate matter increased during the winter relative to the summer (18). In Contra Costa County, these seasonal patterns probably result from meteorological variations, not source differences. Higher concentrations of total suspended particulate matter and other particulate pollutantsare also ienerallg observed during the winter months when rrgional \.entilation is lowest. A second conclusion from this work is that the annual averaee levels of POM mutagens and BAP did not change very muph over the two vear ~.e r i o d. Julv . . 1984-June 1986. This is valuable baseline information for future comparisons.
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Conclusions In this discussion we have attempted to show how the Ames test can assist scientists and managers in the evaluation of some air toxics problems related to mutagens and carcinogens. In the air pollution studies described, the Ames test was applied as a screening tool, not a regulatory device, for air carcinoeen assessment. The test was used to measure the temporal and geographic distributions of airborne muta-eens.. and the results were inteerated into an e~idemioloeical cancer study. It was also used to study pollution sources as well as to follow trends in community air mutagen levels. An important application for the future is in the hioassaydirected fractionation of mutagens in complex air mixtures. Ames testing of chemical fractions will permit more efficient
isolation and identification of hitherto unknown chemical mutagens in air. There is much more work to be done in this area because the specific chemicals identified so far account for only a small fraction of the mutagenicity observed. In future research, this approach will be most useful in "accounting" for the mutagenicity of air samples, that is, determining the percentage of the total mutagenicity caused by the presence of specific chemicals. This "mutagenicity balance" would be analogous to the "mass balance" previously achieved for total suspended particulate matter by air pollution researchers. Acknowledgment Portions of A.I.H.L.'s research was supported by the US EPA and the California Air Resources Board (CARB). The collaboration of CARB Staff Norman Kado and Charles Unger is gratefully acknowledged. The authors also wish to thank Dario Levaggi and Wayman Sui of the Bay Area Air Quality Management, who provided many of the air samples.
Literature Clted 1. Mamn,D.M.;Ames,B. N. Murot. Rrs. 1983.113,173. 2. ~ a e ~ i ~ ~E.:~~gn ne ersA,: ,. Suigerwaid, B.: Thomson. V."Tho Air Toxicn Problem in the United Stater: An Analysis uf Cancer Risks to Public Health for Selected Pollutant.": EPA 45011~85~WL: EPA: Washinson, OC,1985. 3. Dol1.R.; Pe1o.R. J.Nai. Cancer I n r l 198l,66,1193. J. A ; M O I ~ " ~M~ .T. ln ~eiosois:Farmorion and Rmctiuity: 4. ~ a i * ~J., M.; Proceedingsofthe Semnd International Aerosol Conforenee. WestBerlin,Sept. 2226.1986; Pergamon: Olfmd. 1986. 5. Sexton, K.: Wesulowiki. J. J. Enuirun. Sci. Tech. 1985.4.305. 6. MeCsnn, JHospita! Piorlice 1983.18 (91.73. 7. Hsrtman, P. E.: Aukermnn. S. L. In Mmhonirms "/DNA Domlrge and Rwnir; Simie, M. G.: Grossman. L.; Upton, A. C., Fds; Plenum: New York, 1986: pp 407424. 8. Finlayson-Pitt., B. J.: Pitfs. J. N. Atrnosphriir Chemistry; Wiley: New York. 1986:pp 928-935. 9. Kamenr, R.: Bell. D.; Dietrich. A,; Perry. J.; Goodman, R.: Clsrton. L.; Tejada, S. Environ. Sci. Tech. 1985.19.63. 10. Wesolowski, J. J.; Fiesee!, C. P.: Twiss, S.: Chong, J.: Chan. R.; Garcia, L.; Ondo, J.: Fan& A : Lum.S. J.AerosolSci. 1981.12. 208. 11. Auatin. D. F.: Netson. V.; Swain. B.:Johnson. L.; Lum. S.; FI~aaei,P. "Epidemiological Study ofthe Incidence of Cancer as Related to Industrial Ernisions in Contra Costa NC, 19s4. County.California": EPA-6W/Si-84-W8;EP*:ResearchTrianglePark. 12. Kier.L.; Yamssaki,E.:Ames. B. N.Pioc.Nai1. Acod Sei. USA 1974.71.4159. 13. Flessel.C.P.;Gui~uis.G.N.;Cheng,J.C.;Chang.K.;Hahn,E.;Twirs,S.; Wesolowski. ~ . ~ n u i r o n . l n t p r n o1985.11. r. 293. 14. Kado, N. Y.; Guirguis, G. N.; Flersel. C. P.; Chan, R. C.; Chang. K.; Werolownki, J. Enuirnn. Mutogenasis 1986.8,53. IS. Kado, N. Y.: Langley.D.: Eirenstadl.E. Mutol.Rer. 1983,157,227. 16. Pic?. J. N.: Sweetman, J. A,: Harger. N.: Fit% D.: Paw. H. R.: Winer. A. M. J . Air Pollul. Control Amoc. L985,35,638. 17. Gibson.T. J . AirPoIluf. Conlrnl Assoe. 1986.36.1022. I S . Lioy, P. J.; Dsi$ey,.l. M. J . Air Poilvl. Control Assoi. 1983,33,649.
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