75
Mutation Research, 261 (1991) 75-82
© 1991 Elsevier Science Publishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100133Q MUTGEN 01697
Measuring personal exposure to airborne mutagens and nicotine in environmental tobacco smoke Norman S. K a t h a r i n e
Y. Kado
Hammond
a,b, S t e p h e n
A. McCurdy
c, S t e v e n J. T e s l u k
d, D e n n i s P . H . H s i e h b, J e f f J o n e s
b
e a n d M a r c B. S c h e n k e r
c
Research Dicision, California Air Resources Board, Sacramento, CA, b Department of Em,ironmental Toxicology, UniL'ersity of California, Da~,is, CA, c DiL'ision of Occupational and Encironmental Medicine, Department of Internal Medicine, UniL'ersity of California, Dat is, CA, a Department of Family and Community Medicine, Unicersity of Massachusetts Medical School, Worcester, MA and e FPE Group, Lafayette, CA (U.S.A.)
(Received 13 August 1990) (Revision received 4 April 1991) (Accepted 5 April 1991)
Keywords: Personal exposure; Airborne mutagens; Microsuspension assay; Environmental tobacco smoke; Airborne nicotine
Summary
The exposure of individuals to environmental tobacco smoke (ETS) is of increasing public health concern because epidemiological studies have associated passive smoking with increased risk of a variety of adverse health effects among non-smokers including lung cancer. As a way to measure individual exposure to the mutagenic compounds in the complex mixture of ETS, we used a sensitive S a l m o n e l l a / microsome micro pre-incubation (microsuspension) assay to detect mutagenicity of particulate matter collected on filters from low volume (1.7 l / m i n flow rate) personal sampling pumps. Airborne nicotine was collected concurrently as a marker for ETS exposure. In pilot-field studies, individual exposure to ETS was measured in two separate indoor environments in which smokers were present: a gambling casino and a bingo parlor. Total suspended particulate matter (TSP) was collected on filters worn near the breathing zone of non-smoking individuals. Sampling times ranged from 40 min to 6 h. All extracts of filters had detectable levels o f mutagenic activity (TA98, + $9) resulting in airborne mutagenic activity concentrations of 500-5000 r e v / m 3. The mutagenic activity of the filters from the casino and bingo parlors was significantly correlated with total particulate matter per filter (n = 12; Rho = 0.85, p < 0.01) and with airborne nicotine per filter (n = 12; Rho = 0.95, p < 0.0l). The microsuspension assay was sufficiently sensitive to detect the mutagens associated with extracts of particulate matter from low volume samples (0.2-0.6 m 3) in these indoor environments over a relatively short sampling time, and
The results and statements are of the authors and not the California Air Resources Board.
Correspondence: Dr. Norman Y. Kado, California Air Re= sources Board, 1800 15th Street, P.O. Box 2815, Sacramento, CA 95812 (U.S.A.).
76 could be useful in studies of personal exposure to the mutagens in environmental tobacco smoke. Further, airborne nicotine concentrations were highly correlated with airborne mutagenicity and the mutagenic activity associated with ETS could therefore be estimated by the concentrations of nicotine.
Environmental tobacco smoke (ETS) is a complex mixture of gases and particles from burning cigarettes or other tobacco products. Environmental tobacco smoke is composed of aged mainstream and sidestream smoke; where mainstream smoke refers to smoke inhaled by smokers, much of which is exhaled into the environment, and sidestream smoke refers to smoke which is emitted from a burning cigarette or other tobacco product. Exposure of non-smokers to ETS has gained increased public health interest due to epidemiological studies that have found an association of lung cancer and ETS exposure (Hirayama, 1981; National Research Council (NRC), 1986; U.S. Surgeon General, 1986). An important characteristic of ETS is that numerous carcinogenic compounds found in mainstream tobacco smoke are present in greater amounts in sidestream smoke based on mass yields per cigarette (NRC, 1986). Exposure of non-smokers to ETS has most commonly been estimated in epidemiological studies using questionnaires with simple categorical outcomes. A few studies have attempted to quantify the number of smokers in the household and the number of cigarettes consumed. However, such questionnaires may not account for the ventilation characteristics (such as the number of air exchanges) in the house, the dilution volume of the house, individual habits of smokers and non-smokers, and exposures outside the home. Airborne particulate matter found outdoors is generally collected by high volume sampler and its associated mutagenicity has been used as an index of exposure to the complex mixture of carcinogens adsorbed onto these particles (Tokiwa, 1977; Moller and Alfheim, 1980). However, the collection of particles indoors is usually done by medium or low volume sampling pumps to allow collection of personal samples and to minimize disturbance to the normal flow of air within a room. Extracts of the particulate matter collected from ETS have been reported to be
mutagenic in Salmonella (Houdt et al., 1984; I_ofroth and Lazaridis, 1986; Husgafvel-Pursiainen, 1986; Sorsa and Lofroth, 1989) and measurements of mutagenic activity may provide an estimate of individual exposure to mutagenic compounds in the complex mixture of ETS, especially if measurements are obtained from personal sampling. Measurement of personal exposure to airborne mutagenic activity using lowvolume sampling pumps has been difficult due to the absolute amounts of mutagens in particulate matter needed for the Salmonella standard plate-incorporation assay of Ames et al. (1975). Personal sampling measurements are important to more accurately determine individual exposure to mutagens from a variety of sources, including exposure to ETS, since differences in individual habits throughout the day of both the non-smoker and smoker could markedly affect individual exposure to ETS. Since exposure to airborne mutagens may not be entirely attributable to ETS, it is important to examine specific ETS markers in conjunction with mutagenicity. Airborne nicotine has been suggested as one specific marker for ETS exposure (NRC, 1986; Hammond et al., 1987) and provides a convenient indicator of ETS generation. We have previously reported a simple modification (Kado et al., 1983, 1986) of the Salmonella liquid-incubation assay that is at least 10-20 times more sensitive than the plate-incorporation test based on the absolute amount of material required for a specific mutagenic response. Some reports of mutagenicity of low volume filter samples using the microsuspension procedure have recently been published (Kado et al., 1987; Lofroth et al., 1987, 1989). We report here the use of the microsuspension assay for measuring mutagenicity of organic solvent extracts of personal sampling filters. The filters were placed near the breathing zone and connected to low volume sampling pumps worn by individuals exposed to ETS. These portable
77 sampling pumps are worn on the waist and are used widely in occupational exposure studies. These portable units were also placed at a single location (fixed-site) throughout the sampling period. In a pilot study, we examined two separate indoor environments for exposure to the mutagens present in ETS. The mutagenic activity was compared to levels of airborne particulate matter and to airborne nicotine, a marker of ETS. Materials
and
methods
Study subjects and indoor sites Separate groups of non-smoking individuals were studied at two different sampling sites. The first group consisted of 5 volunteers who were stationed at different locations within a gambling casino for approximately 8 h. The casino was approximately 100 × 100 feet (width × length), had a ceiling height of approximately 12 feet, and was carpetted throughout. The room ventilation felt constant and there were few signs of a 'smoky' environment (poor visibility, odor, irritation of the throat, for example). The volunteers walked around within their specific locations in the casino. The approximate number of people in the casino ranged from 95 to 330 based on an hourly count, and of these, approximately 12% were smoking. The second group consisted of 5 volunteers who were located inside a bingo parlor for approximately 4 - 5 h during a single evening. The bingo parlor was approximately 100 x 150 feet (width X length) with a ceiling height of approximately 16 feet with a tile or linoleum floor. There were 377 players counted during the sampling period and approximately 25% of these players were smoking. The volunteers were positioned at specific areas in the bingo parlor. At each location there was placed a 'fixed-site' personal sampiing unit that was identical to the units worn by volunteers. These fixed-site units were placed in a central location relative to the location of the volunteers. There was a distinctly 'smoky' atmosphere.
Air sampling and filter extraction Total particulate matter (TSP) was collected on pre-washed 37-ram Teflon-coated glass fiber
filters (Emfab TX40 HI20WW; Pallflex Corp., Putnam, CT). The filters were pre-washed with dichloromethane (DCM) and dried under nitrogen before use. A second Teflon-coated glass fiber filter that was impregnated with sodium bisulfate was placed behind the first and was used to collect volatile nicotine as described by H a m m o n d et al. (1987). Air was drawn through the filter apparatus with personal sampling pumps (Dupont Instruments, Wilmington, DE) at a flow rate of 1.7 l / m i n . The pumps are designed for maintaining a constant and self-compensating flow rate. Flow rates of the pumps were measured before and immediately after collection. Filters used for trapping of particles were weighed before and after sampling using a microbalance (Cahn Instruments, Inc., Cerritos, CA) at a t e m p e r a t u r e range of 22-25 ° C and at 50% relative humidity. After sampling, each filter was stored in either a Teflon-coated canister or in its collection cassette (plugged at each end) at - 20 ° C until extraction. The filters were cut into quarters with a pre-cleaned razor on glycine paper and placed into pre-cleaned scintillation vials fitted with Teflon-lined screw caps. Dichloromethane (5 ml resi-analysis grade) was added to each vial, the vial was filled with nitrogen and the solution was sonicated for 10 min. The extraction procedure was repeated, the D C M was pooled from the two extractions, evaporated under a stream of nitrogen to dryness and resuspended in D M S O before mutagenicity testing. 5 /zl of the extract were added per incubation tube, which were mathematically converted to cubic meter equivalents added per plate for the d o s e - r e sponse analyses. Blank filters were handled, extracted, and tested in an identical manner as test filters and revertant values from the blanks were subtracted as background activity from all test filter values. All procedures were carried out in a room fitted with yellow lights to minimize photooxidation.
Mutagenicity testing The mutagenic activity of the filter extracts was tested using a Salmonella microsuspension assay previously described (Kado et al., 1983, 1986). The assay is a simple modification of the Salmonella liquid incubation assay of Yahagi et
78 al. (1975). For the microsuspension assay, 0.1 ml of $9 solution or buffer (PBS, 0.15 M, p H 7.4), 0.1 ml of concentrated bacteria, and 0.005 ml of mutagen (filter extract) are added to a 12 × 75 mm glass culture tube. The mixture is incubated for 90 rain at 37 ° C and molten top agar is added to each tube. The top agar-incubation mix solution is poured onto a standard V o g e l - B o n n e r bottom agar as described by Ames et al. (1975). Bacterial tester strain TA98 and $9 (54.5 mg p r o t e i n / m l ) prepared form the Aroclor-induced livers of male S p r a g u e - D a w l e y rats were used throughout. There was 300 Izg of $9 protein per ml mix. Due to the small amount of particulate matter collected on a personal sampling filter, mutagenic activity was determined from triplicate plates and revertants per filter were calculated based on the revertants per plate (minus blank values) multiplied by the dilution factor for dissolving the extract. Data were entered into a V A X 11/750 computer and analyzed using procedures available in the Statistical Analysis System (SAS) software library (SAS Institute, 1985). Spearman's rank correlation coefficient was employed to provide a non-parametric measure of correlation. Results Mutagenic activity from extracts of particulate matter on personal filters could be detected in all personal filter samples tested. The range of mutagenic activity for the filters from the casino and
bingo environments was approximately 500-4000 net r e v e r t a n t s / m 3 ( + $9). The bingo parlor location had higher median concentrations of TSP, nicotine and airborne mutagenic activity compared to the casino site (Table 1). The median concentrations of TSP and specific mutagenic activity for the bingo parlor were 482 / x g / m 3 and 2835 r e v / m 3, respectively, which were over twice the median concentrations of the casino site (200 t z g / m 3 and 1034 rev/m3). The median nicotine concentration at the bingo parlor was 66 / x g / m 3, which was approximately 8 times greater than the concentration measured in the casino. Median nicotine concentration per microgram of TSP in the bingo parlor was approximately 4 times greater than that from the casino; mutagenic activity per microgram of TSP was similar for the casino and bingo sites. The data from both study locations were analyzed first on a per-cubic-meter of air basis and combined for statistical analyses (Fig. 1). The Spearman rank correlation for particulate matter concentration (TSP) and airborne revertant numbers was 0.81 (n = 12, p < 0.001; Fig. la) and the correlation for airborne nicotine concentrations and airborne revertants was 0.53 (n = 12, p < 0.05; Fig. lb). For the nicotine and mutagenicity analysis, the correlation improved considerably when the single outlying point with low airborne nicotine, but high mutagenicity (from the bingo location; Fig. lb) is removed (n = 11, Rho = 0.81). The level of mutagenic activity ( r e v / m 3) predicted by extrapolation to the zero concentration
TABLE 1 MEDIAN AND RANGE OF CONCENTRATIONS OF AIRBORNE PARTICULATE MATTER, NICOTINE AND MUTAGENICITY FOR CASINO AND BINGO PARLOR LOCATIONS Location Casino Site (n = 6)
Median
Bingo Site (n = 6)
Median
Particulate matter (/xg/m3)
Nicotine (/zg/m 3)
Nic./Part. a (Izg//xg)
200 (173-245)
8.02 (3.3-11.6)
0.04
1034 (554-1218)
482 (348-526)
65.5 (4.4-85.4)
0.14
2835 (1919-3 623)
(TA98 rev./tzg) 5.2
Range
Range
/zg nicotine/izg of particulate matter. b TA98 revertants per/xg of particulate matter. a
Mutagenicity (TA98 rev./m 3)
5.9
79 4000
(~
(a)
3000
t-
a) >
2000
o
Casino
•
B i n g o Parlor
rr"
o o oo cr)
o
1000
o
,< I-
•
00
,
,
i
200
,
300
i
•
400
i
,
500
600
Airborne Particulate Matter (~.g/m3)
ters measured were divided by cubic meters of air which introduces variability, we also examined the correlations of absolute levels of particulate matter, nicotine and revertants on a per-filter basis. The correlation of TSP and revertants per filter are presented in Fig. 2a. The Spearman rank correlation was 0.85 ( p < 0.005), which was similar to the value found for the correlation on a per-cubic-meter basis. Although a variety of sampling times are represented in the data presented in Fig. 2a, the best-fit line (y = 5.14x + 25.80) crosses the y axis at 25 revertants/filter, or a
1200"
(a/
4000" (b)
lOOO,
®
E 3000. 800'
o
Casino
•
Bingo P a r b r
O
Casino
•
Bingo Parlor
t-
>
2000-
a
Casino
•
B i n g o Parlor
600'
[]
D
[]
o
n,400' 1000-
1-200'
°@
I.-
© o
oo
0 0
•
•
. 20
•
.
i 4O
.
•
i 6O
.
•
i 80
•
•
5'0
,
i
100
150
200
100 ~ag
Particulate
Matter
/ Filter
Airborne Nicotine (pg/m3)
Fig. 1. (a) Correlation of airborne total particulate matter and mutagenicity per cubic meter of air sampled (Rho = 0.81; Spearman rank; n = 12). (b) Correlation of airborne nicotine and mutagenicity per cubic meter of air sampled at the casino and bingo parlor locations (Rho = 0.53; Spearman rank; n = 12). Circled data points are fixed-site area samples (using personal sampling pumps).
1200 (b)
®
1000
800
6OO
of nicotine is approximately 700 r e v / m 3, a level which may indicate a 'baseline' of indoor mutagenicity. Airborne particulate matter (TSP) in the two study environments appeared to predict the level of mutagenic activity. The slope of the best fit straight line was 7 revertants per microgram of particulate matter; and based on the line, the concentration of particulate matter which represented zero number of revertants expected in the casino and bingo environments was about 100 / ~ g / m 3. However, since the number of data points was limited (n = 12), and the levels of all parame-
[]
400-
D
® o
2OO
0
,
i 5
,
i 10
•
t 15
,
i 20
,
J 25
,
30
I~g Nicotine / Filter
Fig. 2. (a) Correlation of airborne total particulate matter and mutagenicity per filter at the casino and bingo locations (Rho = 0.85, Spearman rank; n = 12). (b) Correlation of airborne nicotine and mutagenicity per filter at the casino and bingo locations (Rho = 0.95, Spearman rank; n = 12). Circled data points are fixed-site area samples (using personal sampling pumps).
80 TABLE 2 SPEARMAN RANK C O R R E L A T I O N VALUES F O R AIRBORNE T O T A L SUSPENDED P A R T I C U L A T E MATTER (TSP), N I C O T I N E AND M U T A G E N I C I T Y
Nicotine/rn 3 Rev./m 3
Nicotine/filter Rev./filter
TSP/m 3
Rev/rn 3
0.54 0.81
0.53 -
TSP/Filter
Rev/Filter
0.87 0.85
0.95 -
n = 12 pairs for all correlations.
level of revertants equal to the spontaneous number of revertants. The correlation of nicotine and revertants per filter are presented in Fig. 2b. The Spearman rank correlation was 0.95 ( p
Mutation Research, 261 (1991) 75-82
© 1991 Elsevier Science Publishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100133Q MUTGEN 01697
Measuring personal exposure to airborne mutagens and nicotine in environmental tobacco smoke Norman S. K a t h a r i n e
Y. Kado
Hammond
a,b, S t e p h e n
A. McCurdy
c, S t e v e n J. T e s l u k
d, D e n n i s P . H . H s i e h b, J e f f J o n e s
b
e a n d M a r c B. S c h e n k e r
c
Research Dicision, California Air Resources Board, Sacramento, CA, b Department of Em,ironmental Toxicology, UniL'ersity of California, Da~,is, CA, c DiL'ision of Occupational and Encironmental Medicine, Department of Internal Medicine, UniL'ersity of California, Dat is, CA, a Department of Family and Community Medicine, Unicersity of Massachusetts Medical School, Worcester, MA and e FPE Group, Lafayette, CA (U.S.A.)
(Received 13 August 1990) (Revision received 4 April 1991) (Accepted 5 April 1991)
Keywords: Personal exposure; Airborne mutagens; Microsuspension assay; Environmental tobacco smoke; Airborne nicotine
Summary
The exposure of individuals to environmental tobacco smoke (ETS) is of increasing public health concern because epidemiological studies have associated passive smoking with increased risk of a variety of adverse health effects among non-smokers including lung cancer. As a way to measure individual exposure to the mutagenic compounds in the complex mixture of ETS, we used a sensitive S a l m o n e l l a / microsome micro pre-incubation (microsuspension) assay to detect mutagenicity of particulate matter collected on filters from low volume (1.7 l / m i n flow rate) personal sampling pumps. Airborne nicotine was collected concurrently as a marker for ETS exposure. In pilot-field studies, individual exposure to ETS was measured in two separate indoor environments in which smokers were present: a gambling casino and a bingo parlor. Total suspended particulate matter (TSP) was collected on filters worn near the breathing zone of non-smoking individuals. Sampling times ranged from 40 min to 6 h. All extracts of filters had detectable levels o f mutagenic activity (TA98, + $9) resulting in airborne mutagenic activity concentrations of 500-5000 r e v / m 3. The mutagenic activity of the filters from the casino and bingo parlors was significantly correlated with total particulate matter per filter (n = 12; Rho = 0.85, p < 0.01) and with airborne nicotine per filter (n = 12; Rho = 0.95, p < 0.0l). The microsuspension assay was sufficiently sensitive to detect the mutagens associated with extracts of particulate matter from low volume samples (0.2-0.6 m 3) in these indoor environments over a relatively short sampling time, and
The results and statements are of the authors and not the California Air Resources Board.
Correspondence: Dr. Norman Y. Kado, California Air Re= sources Board, 1800 15th Street, P.O. Box 2815, Sacramento, CA 95812 (U.S.A.).
76 could be useful in studies of personal exposure to the mutagens in environmental tobacco smoke. Further, airborne nicotine concentrations were highly correlated with airborne mutagenicity and the mutagenic activity associated with ETS could therefore be estimated by the concentrations of nicotine.
Environmental tobacco smoke (ETS) is a complex mixture of gases and particles from burning cigarettes or other tobacco products. Environmental tobacco smoke is composed of aged mainstream and sidestream smoke; where mainstream smoke refers to smoke inhaled by smokers, much of which is exhaled into the environment, and sidestream smoke refers to smoke which is emitted from a burning cigarette or other tobacco product. Exposure of non-smokers to ETS has gained increased public health interest due to epidemiological studies that have found an association of lung cancer and ETS exposure (Hirayama, 1981; National Research Council (NRC), 1986; U.S. Surgeon General, 1986). An important characteristic of ETS is that numerous carcinogenic compounds found in mainstream tobacco smoke are present in greater amounts in sidestream smoke based on mass yields per cigarette (NRC, 1986). Exposure of non-smokers to ETS has most commonly been estimated in epidemiological studies using questionnaires with simple categorical outcomes. A few studies have attempted to quantify the number of smokers in the household and the number of cigarettes consumed. However, such questionnaires may not account for the ventilation characteristics (such as the number of air exchanges) in the house, the dilution volume of the house, individual habits of smokers and non-smokers, and exposures outside the home. Airborne particulate matter found outdoors is generally collected by high volume sampler and its associated mutagenicity has been used as an index of exposure to the complex mixture of carcinogens adsorbed onto these particles (Tokiwa, 1977; Moller and Alfheim, 1980). However, the collection of particles indoors is usually done by medium or low volume sampling pumps to allow collection of personal samples and to minimize disturbance to the normal flow of air within a room. Extracts of the particulate matter collected from ETS have been reported to be
mutagenic in Salmonella (Houdt et al., 1984; I_ofroth and Lazaridis, 1986; Husgafvel-Pursiainen, 1986; Sorsa and Lofroth, 1989) and measurements of mutagenic activity may provide an estimate of individual exposure to mutagenic compounds in the complex mixture of ETS, especially if measurements are obtained from personal sampling. Measurement of personal exposure to airborne mutagenic activity using lowvolume sampling pumps has been difficult due to the absolute amounts of mutagens in particulate matter needed for the Salmonella standard plate-incorporation assay of Ames et al. (1975). Personal sampling measurements are important to more accurately determine individual exposure to mutagens from a variety of sources, including exposure to ETS, since differences in individual habits throughout the day of both the non-smoker and smoker could markedly affect individual exposure to ETS. Since exposure to airborne mutagens may not be entirely attributable to ETS, it is important to examine specific ETS markers in conjunction with mutagenicity. Airborne nicotine has been suggested as one specific marker for ETS exposure (NRC, 1986; Hammond et al., 1987) and provides a convenient indicator of ETS generation. We have previously reported a simple modification (Kado et al., 1983, 1986) of the Salmonella liquid-incubation assay that is at least 10-20 times more sensitive than the plate-incorporation test based on the absolute amount of material required for a specific mutagenic response. Some reports of mutagenicity of low volume filter samples using the microsuspension procedure have recently been published (Kado et al., 1987; Lofroth et al., 1987, 1989). We report here the use of the microsuspension assay for measuring mutagenicity of organic solvent extracts of personal sampling filters. The filters were placed near the breathing zone and connected to low volume sampling pumps worn by individuals exposed to ETS. These portable
77 sampling pumps are worn on the waist and are used widely in occupational exposure studies. These portable units were also placed at a single location (fixed-site) throughout the sampling period. In a pilot study, we examined two separate indoor environments for exposure to the mutagens present in ETS. The mutagenic activity was compared to levels of airborne particulate matter and to airborne nicotine, a marker of ETS. Materials
and
methods
Study subjects and indoor sites Separate groups of non-smoking individuals were studied at two different sampling sites. The first group consisted of 5 volunteers who were stationed at different locations within a gambling casino for approximately 8 h. The casino was approximately 100 × 100 feet (width × length), had a ceiling height of approximately 12 feet, and was carpetted throughout. The room ventilation felt constant and there were few signs of a 'smoky' environment (poor visibility, odor, irritation of the throat, for example). The volunteers walked around within their specific locations in the casino. The approximate number of people in the casino ranged from 95 to 330 based on an hourly count, and of these, approximately 12% were smoking. The second group consisted of 5 volunteers who were located inside a bingo parlor for approximately 4 - 5 h during a single evening. The bingo parlor was approximately 100 x 150 feet (width X length) with a ceiling height of approximately 16 feet with a tile or linoleum floor. There were 377 players counted during the sampling period and approximately 25% of these players were smoking. The volunteers were positioned at specific areas in the bingo parlor. At each location there was placed a 'fixed-site' personal sampiing unit that was identical to the units worn by volunteers. These fixed-site units were placed in a central location relative to the location of the volunteers. There was a distinctly 'smoky' atmosphere.
Air sampling and filter extraction Total particulate matter (TSP) was collected on pre-washed 37-ram Teflon-coated glass fiber
filters (Emfab TX40 HI20WW; Pallflex Corp., Putnam, CT). The filters were pre-washed with dichloromethane (DCM) and dried under nitrogen before use. A second Teflon-coated glass fiber filter that was impregnated with sodium bisulfate was placed behind the first and was used to collect volatile nicotine as described by H a m m o n d et al. (1987). Air was drawn through the filter apparatus with personal sampling pumps (Dupont Instruments, Wilmington, DE) at a flow rate of 1.7 l / m i n . The pumps are designed for maintaining a constant and self-compensating flow rate. Flow rates of the pumps were measured before and immediately after collection. Filters used for trapping of particles were weighed before and after sampling using a microbalance (Cahn Instruments, Inc., Cerritos, CA) at a t e m p e r a t u r e range of 22-25 ° C and at 50% relative humidity. After sampling, each filter was stored in either a Teflon-coated canister or in its collection cassette (plugged at each end) at - 20 ° C until extraction. The filters were cut into quarters with a pre-cleaned razor on glycine paper and placed into pre-cleaned scintillation vials fitted with Teflon-lined screw caps. Dichloromethane (5 ml resi-analysis grade) was added to each vial, the vial was filled with nitrogen and the solution was sonicated for 10 min. The extraction procedure was repeated, the D C M was pooled from the two extractions, evaporated under a stream of nitrogen to dryness and resuspended in D M S O before mutagenicity testing. 5 /zl of the extract were added per incubation tube, which were mathematically converted to cubic meter equivalents added per plate for the d o s e - r e sponse analyses. Blank filters were handled, extracted, and tested in an identical manner as test filters and revertant values from the blanks were subtracted as background activity from all test filter values. All procedures were carried out in a room fitted with yellow lights to minimize photooxidation.
Mutagenicity testing The mutagenic activity of the filter extracts was tested using a Salmonella microsuspension assay previously described (Kado et al., 1983, 1986). The assay is a simple modification of the Salmonella liquid incubation assay of Yahagi et
78 al. (1975). For the microsuspension assay, 0.1 ml of $9 solution or buffer (PBS, 0.15 M, p H 7.4), 0.1 ml of concentrated bacteria, and 0.005 ml of mutagen (filter extract) are added to a 12 × 75 mm glass culture tube. The mixture is incubated for 90 rain at 37 ° C and molten top agar is added to each tube. The top agar-incubation mix solution is poured onto a standard V o g e l - B o n n e r bottom agar as described by Ames et al. (1975). Bacterial tester strain TA98 and $9 (54.5 mg p r o t e i n / m l ) prepared form the Aroclor-induced livers of male S p r a g u e - D a w l e y rats were used throughout. There was 300 Izg of $9 protein per ml mix. Due to the small amount of particulate matter collected on a personal sampling filter, mutagenic activity was determined from triplicate plates and revertants per filter were calculated based on the revertants per plate (minus blank values) multiplied by the dilution factor for dissolving the extract. Data were entered into a V A X 11/750 computer and analyzed using procedures available in the Statistical Analysis System (SAS) software library (SAS Institute, 1985). Spearman's rank correlation coefficient was employed to provide a non-parametric measure of correlation. Results Mutagenic activity from extracts of particulate matter on personal filters could be detected in all personal filter samples tested. The range of mutagenic activity for the filters from the casino and
bingo environments was approximately 500-4000 net r e v e r t a n t s / m 3 ( + $9). The bingo parlor location had higher median concentrations of TSP, nicotine and airborne mutagenic activity compared to the casino site (Table 1). The median concentrations of TSP and specific mutagenic activity for the bingo parlor were 482 / x g / m 3 and 2835 r e v / m 3, respectively, which were over twice the median concentrations of the casino site (200 t z g / m 3 and 1034 rev/m3). The median nicotine concentration at the bingo parlor was 66 / x g / m 3, which was approximately 8 times greater than the concentration measured in the casino. Median nicotine concentration per microgram of TSP in the bingo parlor was approximately 4 times greater than that from the casino; mutagenic activity per microgram of TSP was similar for the casino and bingo sites. The data from both study locations were analyzed first on a per-cubic-meter of air basis and combined for statistical analyses (Fig. 1). The Spearman rank correlation for particulate matter concentration (TSP) and airborne revertant numbers was 0.81 (n = 12, p < 0.001; Fig. la) and the correlation for airborne nicotine concentrations and airborne revertants was 0.53 (n = 12, p < 0.05; Fig. lb). For the nicotine and mutagenicity analysis, the correlation improved considerably when the single outlying point with low airborne nicotine, but high mutagenicity (from the bingo location; Fig. lb) is removed (n = 11, Rho = 0.81). The level of mutagenic activity ( r e v / m 3) predicted by extrapolation to the zero concentration
TABLE 1 MEDIAN AND RANGE OF CONCENTRATIONS OF AIRBORNE PARTICULATE MATTER, NICOTINE AND MUTAGENICITY FOR CASINO AND BINGO PARLOR LOCATIONS Location Casino Site (n = 6)
Median
Bingo Site (n = 6)
Median
Particulate matter (/xg/m3)
Nicotine (/zg/m 3)
Nic./Part. a (Izg//xg)
200 (173-245)
8.02 (3.3-11.6)
0.04
1034 (554-1218)
482 (348-526)
65.5 (4.4-85.4)
0.14
2835 (1919-3 623)
(TA98 rev./tzg) 5.2
Range
Range
/zg nicotine/izg of particulate matter. b TA98 revertants per/xg of particulate matter. a
Mutagenicity (TA98 rev./m 3)
5.9
79 4000
(~
(a)
3000
t-
a) >
2000
o
Casino
•
B i n g o Parlor
rr"
o o oo cr)
o
1000
o
,< I-
•
00
,
,
i
200
,
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ters measured were divided by cubic meters of air which introduces variability, we also examined the correlations of absolute levels of particulate matter, nicotine and revertants on a per-filter basis. The correlation of TSP and revertants per filter are presented in Fig. 2a. The Spearman rank correlation was 0.85 ( p < 0.005), which was similar to the value found for the correlation on a per-cubic-meter basis. Although a variety of sampling times are represented in the data presented in Fig. 2a, the best-fit line (y = 5.14x + 25.80) crosses the y axis at 25 revertants/filter, or a
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Particulate
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Airborne Nicotine (pg/m3)
Fig. 1. (a) Correlation of airborne total particulate matter and mutagenicity per cubic meter of air sampled (Rho = 0.81; Spearman rank; n = 12). (b) Correlation of airborne nicotine and mutagenicity per cubic meter of air sampled at the casino and bingo parlor locations (Rho = 0.53; Spearman rank; n = 12). Circled data points are fixed-site area samples (using personal sampling pumps).
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of nicotine is approximately 700 r e v / m 3, a level which may indicate a 'baseline' of indoor mutagenicity. Airborne particulate matter (TSP) in the two study environments appeared to predict the level of mutagenic activity. The slope of the best fit straight line was 7 revertants per microgram of particulate matter; and based on the line, the concentration of particulate matter which represented zero number of revertants expected in the casino and bingo environments was about 100 / ~ g / m 3. However, since the number of data points was limited (n = 12), and the levels of all parame-
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Fig. 2. (a) Correlation of airborne total particulate matter and mutagenicity per filter at the casino and bingo locations (Rho = 0.85, Spearman rank; n = 12). (b) Correlation of airborne nicotine and mutagenicity per filter at the casino and bingo locations (Rho = 0.95, Spearman rank; n = 12). Circled data points are fixed-site area samples (using personal sampling pumps).
80 TABLE 2 SPEARMAN RANK C O R R E L A T I O N VALUES F O R AIRBORNE T O T A L SUSPENDED P A R T I C U L A T E MATTER (TSP), N I C O T I N E AND M U T A G E N I C I T Y
Nicotine/rn 3 Rev./m 3
Nicotine/filter Rev./filter
TSP/m 3
Rev/rn 3
0.54 0.81
0.53 -
TSP/Filter
Rev/Filter
0.87 0.85
0.95 -
n = 12 pairs for all correlations.
level of revertants equal to the spontaneous number of revertants. The correlation of nicotine and revertants per filter are presented in Fig. 2b. The Spearman rank correlation was 0.95 ( p
