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Effect of emissions from residential wood stoves on SCE induction in CHO cells
Mutation Research, 118 (1983) 69-75 Elsevier
69
M R 0788
Effect of emissions from residential wood stoves on SCE induction in C H O cells S. H y t 6 n e n 1, I. A l f h e i m 2 and M. Sorsa 1 I Institute of Occupational Health, Helsinki (Finland) and 2 Central Institute for Industrial Research, Oslo
(Norway)
(Received 16 November 1982) (Revisionreceived28 February 1983) (Accepted 9 March 1983)
Summaff The SCE-induction capacity of emissions from an airtight horizontal baffled residential wood stove was investigated in CHO cells. The samples were taken under normal and starved air conditions, from burning birch and spruce separately. Both particle phase and vapour phase were collected. All samples induced a dose-related response in SCE both with and without a metabolic activation system, the rat-liver microsomal fraction. The burning conditions in the stove influenced the mutagenicity of the emissions more than the type of wood; the smoke from wood burning under starved air conditions was more than one order of magnitude more potent in inducing a significant SCE response. With all samples, the response in SCE induction was highest without metabolic activation. The toxicity of the samples, especially those without $9, limited the dose-range tested.
The presence of mutagenic and carcinogenic agents, particularly polycyclic aromatic hydrocarbons (PAH), in combustion emissions is well known (Guerin, 1978). The use of wood in residential heating has a long tradition in the northern countries and, owing to rising oil prices, it is also becoming an increasingly important alternative as a domestic energy source in the future. The mutagenicity of atmospheric pollutants has primarily been tested in the Ames assay (Chrisp and Fisher, 1980). However, for human risk evaluation, it is also important to apply test systems other than bacterial ones. The induction of sisterchromatid exchanges (SCE) is a sensitive indicator of mutagenic carcinogens (Perry,
Reprint requests may be sent to: Dr. M. Sorsa, Institute of Occupational Health, Haartmaninkatu 1, SF-00290 Helsinki 29 (Finland). 0165-1218/83/$03.00/© 1983 Elsevier Science Publishers B.V.
70 1980), and it is a widely used test system for the screening of such compounds. For example, SCE analysis has been used in investigating the genotoxic effects of organic-extractable components of urban airborne particles (Lockard et al., 1981) and for comparing the mutagenic activities of extracts from ambient and forest-fire polluted air (Viau et al., 1982). In this study we used the SCE analysis in Chinese hamster ovary (CHO) cells to detect the genotoxicity of emissions from wood combustion. The same smoke extracts have also been tested with the Salmonella/microsome assay (M~ller et al., 1982; Ramdahl et al., 1982).
Material and methods
Sample collection and preparation A small 'airtight' horizontal baffled residential wood stove, with a typical heat output of 5 - 4 kW, was used for wood burning. Burning under normal conditions involved an air supply setting in front of the stove to ensure burning with flames. Under starved-air conditions the air supply was completely restricted, producing only a faint glow. The wood used was birch or spruce with 15% water content, as determined by drying the sample at 105°C until constant weight was obtained. The flue gas was sampled from the stack with a quartz glass probe 1 m above the stove. The particles in the flue gas were collected on a Gelman glass-fibre filter type A - E , held at 125°C. The flue gas was cooled and the condensate collected in several impingers cooled gradually from 0 to - 6 0 ° C . The dried flue gas was finally passed through a column containing Amberlite XAD-2 adsorbent; the Amberlite had been cleaned by sequential Soxhlet extraction with methanol, diethyl ether, acetone, methanol and acetone, 24 h with each solvent. The filter and XAD-2 were extracted with acetone in a Soxhlet apparatus for 24 h. The condensate was acidified to p H 2 and extracted with dichloromethane. After extraction, the D C M was evaporated and the residue taken up in acetone. The 3 acetone extracts were combined to make a total extract. The samples were analysed for PAH and for other organic compounds. A detailed description of the analytical procedures and of the results has been published elsewhere (Ramdahl et al., 1982). SCE assay The SCE assay was conducted in Chinese hamster ovary (CHO) cells. Cells were grown in 25-cm 2 plastic flasks in McCoy's 5A medium supplemented with 15% foetal calf serum (Gibco), 100 U of penicillin per ml and 100/~g of streptomycin per ml. For tests with and without $9 activation, cultures were initiated with 2.5 x 105 ceils in 5 ml of culture medium and incubated in 4% CO 2 in air at 37°C. After 24 h, when the cells were growing exponentially, the culture medium was replaced with 5 ml of medium without serum but with various concentrations of smoke extracts, acetone or cyclophosphamide. For metabolic activation, 1.5 ml of $9 mix containing 10% $9 was added to each flask (final volume 5 ml).
71 The $9 (containing 25 mg protein/ml) was prepared from male rats induced by Clophen A-50 R (Bayer AG) according to the procedure described by Ames et al. (1975). The $9 mix was prepared as follows 3 ml 20 mM Hepes buffer, 0.15 M KC1 solution, p H 7.4; 1.3 ml 80 mM MgSO4; 2.7 ml McCoy's 5A medium without serum; 1.0 ml 50 mM glucose-6-P; 1.0 ml 40 mM NADP; and 1.0 ml $9 fraction. The CHO cultures were exposed to the test chemicals for 1 h. The cells were then rinsed twice with buffered saline solution (PBS, Gibco), 5 ml of culture medium containing 10/~M BUdR was added to each dish and the cells were incubated for a further 24 h. Colcemid (2 x 10 - 7 M final concentration) was added for the final 2 h. The mitotic cells were collected by shaking and treated with hypotonic solution (0.2 g KC1 and 0.2 g tri-Na-citrate in 90 ml distilled water and 10 ml foetal calf serum, see Miller et al., 1976) for 15 min at 37°C, and then fixed with 3 : 1 methanol : acetic acid. Air-dried preparations were stained by the fluorescence-plus-Giemsa (FPG) technique (Perry and Wolff 1974) to provide permanently stained harlequin chromosomes. By using coded slides, 60 cells from duplicate cultures at each treatment were scored for SCEs. Statistical significances of the results were tested in a 2-tailed Student's t test.
Results and discussion
For combustion emissions it is important to investigate the gas-phase organics in addition to the particle phase commonly collected. PAH and other compounds are not completely condensed on particles in the hot flue gases (Natusch, 1978). Also, evaporative losses from the filters may occur (Pupp et al., 1974). In this study the loss of material was avoided by collecting the condensate and organics adsorbed on XAD-2. The capacity of the samples to induce SCE is shown in Table 1. A dose-response in SCE was obtained for all samples in the presence and absence of $9 mix (Figs. la,b). The toxicity of the samples limited the dose range tested at the highest concentrations; obvious toxic effects like cell death, mitotic delay and chromosome aberrations and fragmentation were observed. As in the results observed for the same samples in the Salmonella assay (Moller et al., 1982), the greatest SCE-inducing activity was observed with the samples from starved-air conditions. These samples also contained the greatest concentration of PAHs (Ramdahl et al., 1982), as a result of incomplete combustion. The mutagenic potency of emissions from starved-air combustion was 10-50-fold greater than that observed under normal burning conditions. Differences between the type of wood were less marked. In CHO cells, the SCE-induction potency was greater in all samples without metabolic activation, while in the Salmonella assay (strain TA98) the addition of $9 gave variable results, increasing or decreasing the mutagenicity depending on the sample (M~ller et al., 1982). The results thus suggest that the samples also contained non-PAH mutagens with direct mutagenic potency. However, the time of exposure (1 h) was relatively short, and it has been shown that some promutagens/pro-
0.8
1.6
1.9
3.2
3.7
6.4
7.4
8.0
14.8
0.016
0.032
0.023
0.063
0.046
0.126
0.092
0.158
0.184
4.0
5.0
2.0
4.0
1.0
2.0
0.5
1.0
0.5
- $9
17.1 _+0.7 ***
16.2 --+0.8 ***
toxic
toxic
12.7 + 0 . 7 ***
9.7 -+0.4 11.4 _+0.5***
9.0 -+0.4 8.9 _+0.4
11.0 _+0.5 *
- $9
+ $9
+ $9
+ $9
~tl/5 ml
1 smoke
mg dry wood
N o r m a l air
Normal air
Amount of sample
Starved air
Spruce wood
Birch w o o d
FROM DUPLICATED
Treatment
I N D U C T I O N O F S C E s (MEAN~CELL _+ SE) I N C H O C E L L S (60 C E L L S A N A L Y S E D FROM WOOD BURNING WITH AIR OR WITH STARVED AIR
TABLE 1
- $9
CULTURES)
10.4 __+0.4 **
11.0 + 0 . 5 **
9.4 +0.4
9.4 _+0.3
+ $9
S t a r v e d air
15.7 + 0 . 7 a***
14.7 + 0 . 6 ***
10.9 +0.5 *
11.5 _+0.4 ***
- $9
BY S M O K E E M I S S I O N S
84
95
105
114
126
0.86
1.05
1.08
1.26
1.29
-+3.3 ***
98 _+0.5
10.3
40
5 × 10 - 5 M
9.8
_+0.6 a***
16.6
.
_+0.7 ***
_+0.4 -
.
9,1
a***
***
_+0.4 -
-
_+0.6
13.2
.
_+0.5
17.1
_+0.5 *** . .
11.8
13.7 _+0.5 *** .
11,2
.
_+0.5 *** .
60
60
60
40
40
25
12.7
.
+ 0 . 6 ** .
.
11.0
.
.
.
.
.
_+5.0 ***
89
_+0.4
8.6
8.8 -+0.4
9.4
-
-
-
-
-
-
-+0.4
-
-
-
-
-
* P < 0.05, ** P < 0.01, *** P < 0.001, 2-tailed t test, c o m p a r e d with c o r r e s p o n d i n g control value. a Toxic effects (cell d e a t h , mitotic delay, c h r o m o s o m e f r a g m e n t a t i o n ) . b Because of toxicity, only 30 cells c o u l d be analysed.
(cyclophosphamide)
Positive control
(acetone)
50
76
0.84
Control
50
53
0.54
25
48
0.53
8.0
29.6
0.368
> 100 ***
10.2 -+0.4
10.8 +0.5
+0.4 *
10.4 _+0.8
10.8 _+0.4
14.1 + 0 . 5 a***
16.4 _+0.6 ***
_+0.5 ***
11.4
13.9
-+0.4
12.9 + 0 . 5 **
11.0
_+0.5
10.1
73 _+8.3 ***
8.8 _+0.4
15.0 4-0.6 a***
8.9 _+0.3
9.4 _+0.4
toxic
74 ,,18 a
ai
*' 1 6 = ®
/
I
18~ •
b
~
.,,6
~4
8 0.4
0.8
1.2
1.6 2.0 2.4 2.8 m g dry wood b u r n e d l m l
6.0 ~
2
6
10
14 18 22 mg dry wood b u r n e d / m l
26
Fig. 1. Induction of SCEs in C H O cells treated with smoke emission samples from a wood stove with (a) starved air combustion or (b) normal air combustion burning birch (circles) or spruce (squares). Experiments were carried out with $9 mix (dark symbols) and without $9 mix (open symbols).
carcinogens may need a longer exposure for their metabolic activation (Takehisa and Wolff, 1978; Nachtman and Wolff, 1982). Moreover, the possibility that the toxicity of the samples had interfered with the activity of the $9 enzymes cannot be excluded. Whereas the addition of $9 mix reduced the toxicity of the samples, it is possible that the enhanced mutagenicity of some samples in the presence of $9 in the Salmonella assay may not represent a true metabolic activation effect but rather an unmasking of the direct mutagenicity in the sample, previously undetected because of high toxicity. Consequently, the lack of an S9-mediated response may have resulted from the experimental conditions and may not accurately reflect the absence or presence of promutagens in the samples. The complexity of the activating and deactivating reactions in mixtures of mutagenic compounds makes it difficult to separate the mutagenicity caused by direct acting compounds from the mutagenicity caused by compounds that do not require $9 microsomal activation. Since the metabolic capacity of CHO cells in activating indirect mutagens is known to be extremely poor (Stetka and Wolff, 1976), the direct mutagenicity observed in all the samples firmly suggests that agents other than PAHs are responsible for the SCE induction. The mutagenicity of emissions from wood combustion has not been widely examined in systems other than the Salmonella assay. Using the Salmonella assay, L6froth (1978) showed that combusion emissions of wood chips did not induce mutagenicity in strain TA1535, indicating that mutagens causing base-pair substitutions were not present in detectable concentrations. The absence of mutagenicity in strain TA1535 and the presence of mutagenicity in the frame-shift detector strains strongly indicated that the mutagenic compounds were mainly polycyclic compounds. The high potency of the smoke emissions to induce SCEs in rodent cells, in addition to the earlier information on their capacity to induce response in the Salmonella/microsome assay, emphasizes the significance of combustion emissions as a source of ambient mutagenicity in the human environment.
75
Acknowledgements The study received financial support from the Nordic Council of Ministers, through the MIL-2 project and a grant from the Ministry of Interior Affairs, Government of Finland. We thank our colleague Dr. Kaija Linnainmaa for help and advice with the CHO cell system.
References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsomemutagenicity test, Mutation Res., 31,347-364. Chrisp, C.E., and G.L. Fisher (1980) Mutagenicity of airborne particles, Mutation Res., 76, 143-164. Guerin, M.R. (1978) Energy sources of polycyclic aromatic hydrocarbons, in: H.V. Gelboin and P.O.P. Ts'o (Eds.), Polycyclic Aromatic Hydrocarbons and Cancer, Vol. 1, Environment, Chemistry and Metabolism, Academic Press, New York, pp. 3-42. Lockard, J.M., C.J. Viau, C. Lee-Stephens, J.C. Caldwell, P. Wojciechowski, H.G. Enoch and P.S. Sabharwal (1981) Induction of sister-chromatid exchanges and bacterial revertants by organic extracts of airborne particles, Environ. Mutagen., 3, 671-681. Lrfroth, G. (1978) Mutagenicity assay of combustion emissions, Chemosphere, 7, 791-798. Miller, R.C., M.M. Aronson and W.W. Nichols (1976) Effects of treatment of differential staining of BrdU labelled metaphase chromosomes: Three-way differentiation of M 3 chromosomes, Chromosoma, 55, 1-11. Moller, M., I. Alfheim and J. Hongslo (1982) The effect of emissions from residential wood stove on the mutagenicity in Salmonella typhimurium, 12th Annual Meeting of EEMS, Espoo, Finland, 20-24 June. Nachtman, J.P., and S. Wolff (1982) Activity of nitro-polynuclear aromatic hydrocarbons in the sister chromatid exchange assay with and without metabolic activation, Environ. Mutagen., 4, 1-5. Natusch, D.F.S. (1978) Potentially carcinogenic species emitted to the atmosphere by fossil-fueled power plants, Environ. Health Persp., 22, 79-90. Perry, P.E. (1980) Chemical mutagens and sister chromatid exchange, in: F.J. de Serres and A. Hollaender (Eds.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 6, Plenum, New York. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature (London), 251, 156-158. Pupp, C., R.C. Lao, J.J. Murray and R.F. Pottie (1974) Equilibrium vapour concentrations of some polycyclic aromatic hydrocarbons, As406 and SeO2 and the collection efficiencies of these air pollutants, Atmos. Environ., 8, 915-925. Ramdahl, T., I. Alfheim, S. Rustad and T. Olsen (1982) Chemical and biological characterization of emissions from small residential stoves burning wood and charcoal, Chemosphere, 11,601-611. Stetka, D.G., and S. Wolff (1976) Sister chromatid exchange as an assay for genetic damage induced by mutagen-carcinogens, II. In vitro test for compounds requiring metabolic activation, Mutation Res., 41,343-350. Takehisa, S., and S. Wolff (1978) The induction of sister chromatid exchanges in Chinese hamster ovary cells by prolonged exposure to 2-acetylaminofluorene and $9 mix, Mutation Res., 58, 103-106. Viau, C.J., J.M. Lockard, H.G. Enoch and P.S. Sabharwal (1982) Comparison of the genotoxic activities of extracts from ambient and forest fire polluted air, Environ. Mutagen., 4, 37-43.
69
M R 0788
Effect of emissions from residential wood stoves on SCE induction in C H O cells S. H y t 6 n e n 1, I. A l f h e i m 2 and M. Sorsa 1 I Institute of Occupational Health, Helsinki (Finland) and 2 Central Institute for Industrial Research, Oslo
(Norway)
(Received 16 November 1982) (Revisionreceived28 February 1983) (Accepted 9 March 1983)
Summaff The SCE-induction capacity of emissions from an airtight horizontal baffled residential wood stove was investigated in CHO cells. The samples were taken under normal and starved air conditions, from burning birch and spruce separately. Both particle phase and vapour phase were collected. All samples induced a dose-related response in SCE both with and without a metabolic activation system, the rat-liver microsomal fraction. The burning conditions in the stove influenced the mutagenicity of the emissions more than the type of wood; the smoke from wood burning under starved air conditions was more than one order of magnitude more potent in inducing a significant SCE response. With all samples, the response in SCE induction was highest without metabolic activation. The toxicity of the samples, especially those without $9, limited the dose-range tested.
The presence of mutagenic and carcinogenic agents, particularly polycyclic aromatic hydrocarbons (PAH), in combustion emissions is well known (Guerin, 1978). The use of wood in residential heating has a long tradition in the northern countries and, owing to rising oil prices, it is also becoming an increasingly important alternative as a domestic energy source in the future. The mutagenicity of atmospheric pollutants has primarily been tested in the Ames assay (Chrisp and Fisher, 1980). However, for human risk evaluation, it is also important to apply test systems other than bacterial ones. The induction of sisterchromatid exchanges (SCE) is a sensitive indicator of mutagenic carcinogens (Perry,
Reprint requests may be sent to: Dr. M. Sorsa, Institute of Occupational Health, Haartmaninkatu 1, SF-00290 Helsinki 29 (Finland). 0165-1218/83/$03.00/© 1983 Elsevier Science Publishers B.V.
70 1980), and it is a widely used test system for the screening of such compounds. For example, SCE analysis has been used in investigating the genotoxic effects of organic-extractable components of urban airborne particles (Lockard et al., 1981) and for comparing the mutagenic activities of extracts from ambient and forest-fire polluted air (Viau et al., 1982). In this study we used the SCE analysis in Chinese hamster ovary (CHO) cells to detect the genotoxicity of emissions from wood combustion. The same smoke extracts have also been tested with the Salmonella/microsome assay (M~ller et al., 1982; Ramdahl et al., 1982).
Material and methods
Sample collection and preparation A small 'airtight' horizontal baffled residential wood stove, with a typical heat output of 5 - 4 kW, was used for wood burning. Burning under normal conditions involved an air supply setting in front of the stove to ensure burning with flames. Under starved-air conditions the air supply was completely restricted, producing only a faint glow. The wood used was birch or spruce with 15% water content, as determined by drying the sample at 105°C until constant weight was obtained. The flue gas was sampled from the stack with a quartz glass probe 1 m above the stove. The particles in the flue gas were collected on a Gelman glass-fibre filter type A - E , held at 125°C. The flue gas was cooled and the condensate collected in several impingers cooled gradually from 0 to - 6 0 ° C . The dried flue gas was finally passed through a column containing Amberlite XAD-2 adsorbent; the Amberlite had been cleaned by sequential Soxhlet extraction with methanol, diethyl ether, acetone, methanol and acetone, 24 h with each solvent. The filter and XAD-2 were extracted with acetone in a Soxhlet apparatus for 24 h. The condensate was acidified to p H 2 and extracted with dichloromethane. After extraction, the D C M was evaporated and the residue taken up in acetone. The 3 acetone extracts were combined to make a total extract. The samples were analysed for PAH and for other organic compounds. A detailed description of the analytical procedures and of the results has been published elsewhere (Ramdahl et al., 1982). SCE assay The SCE assay was conducted in Chinese hamster ovary (CHO) cells. Cells were grown in 25-cm 2 plastic flasks in McCoy's 5A medium supplemented with 15% foetal calf serum (Gibco), 100 U of penicillin per ml and 100/~g of streptomycin per ml. For tests with and without $9 activation, cultures were initiated with 2.5 x 105 ceils in 5 ml of culture medium and incubated in 4% CO 2 in air at 37°C. After 24 h, when the cells were growing exponentially, the culture medium was replaced with 5 ml of medium without serum but with various concentrations of smoke extracts, acetone or cyclophosphamide. For metabolic activation, 1.5 ml of $9 mix containing 10% $9 was added to each flask (final volume 5 ml).
71 The $9 (containing 25 mg protein/ml) was prepared from male rats induced by Clophen A-50 R (Bayer AG) according to the procedure described by Ames et al. (1975). The $9 mix was prepared as follows 3 ml 20 mM Hepes buffer, 0.15 M KC1 solution, p H 7.4; 1.3 ml 80 mM MgSO4; 2.7 ml McCoy's 5A medium without serum; 1.0 ml 50 mM glucose-6-P; 1.0 ml 40 mM NADP; and 1.0 ml $9 fraction. The CHO cultures were exposed to the test chemicals for 1 h. The cells were then rinsed twice with buffered saline solution (PBS, Gibco), 5 ml of culture medium containing 10/~M BUdR was added to each dish and the cells were incubated for a further 24 h. Colcemid (2 x 10 - 7 M final concentration) was added for the final 2 h. The mitotic cells were collected by shaking and treated with hypotonic solution (0.2 g KC1 and 0.2 g tri-Na-citrate in 90 ml distilled water and 10 ml foetal calf serum, see Miller et al., 1976) for 15 min at 37°C, and then fixed with 3 : 1 methanol : acetic acid. Air-dried preparations were stained by the fluorescence-plus-Giemsa (FPG) technique (Perry and Wolff 1974) to provide permanently stained harlequin chromosomes. By using coded slides, 60 cells from duplicate cultures at each treatment were scored for SCEs. Statistical significances of the results were tested in a 2-tailed Student's t test.
Results and discussion
For combustion emissions it is important to investigate the gas-phase organics in addition to the particle phase commonly collected. PAH and other compounds are not completely condensed on particles in the hot flue gases (Natusch, 1978). Also, evaporative losses from the filters may occur (Pupp et al., 1974). In this study the loss of material was avoided by collecting the condensate and organics adsorbed on XAD-2. The capacity of the samples to induce SCE is shown in Table 1. A dose-response in SCE was obtained for all samples in the presence and absence of $9 mix (Figs. la,b). The toxicity of the samples limited the dose range tested at the highest concentrations; obvious toxic effects like cell death, mitotic delay and chromosome aberrations and fragmentation were observed. As in the results observed for the same samples in the Salmonella assay (Moller et al., 1982), the greatest SCE-inducing activity was observed with the samples from starved-air conditions. These samples also contained the greatest concentration of PAHs (Ramdahl et al., 1982), as a result of incomplete combustion. The mutagenic potency of emissions from starved-air combustion was 10-50-fold greater than that observed under normal burning conditions. Differences between the type of wood were less marked. In CHO cells, the SCE-induction potency was greater in all samples without metabolic activation, while in the Salmonella assay (strain TA98) the addition of $9 gave variable results, increasing or decreasing the mutagenicity depending on the sample (M~ller et al., 1982). The results thus suggest that the samples also contained non-PAH mutagens with direct mutagenic potency. However, the time of exposure (1 h) was relatively short, and it has been shown that some promutagens/pro-
0.8
1.6
1.9
3.2
3.7
6.4
7.4
8.0
14.8
0.016
0.032
0.023
0.063
0.046
0.126
0.092
0.158
0.184
4.0
5.0
2.0
4.0
1.0
2.0
0.5
1.0
0.5
- $9
17.1 _+0.7 ***
16.2 --+0.8 ***
toxic
toxic
12.7 + 0 . 7 ***
9.7 -+0.4 11.4 _+0.5***
9.0 -+0.4 8.9 _+0.4
11.0 _+0.5 *
- $9
+ $9
+ $9
+ $9
~tl/5 ml
1 smoke
mg dry wood
N o r m a l air
Normal air
Amount of sample
Starved air
Spruce wood
Birch w o o d
FROM DUPLICATED
Treatment
I N D U C T I O N O F S C E s (MEAN~CELL _+ SE) I N C H O C E L L S (60 C E L L S A N A L Y S E D FROM WOOD BURNING WITH AIR OR WITH STARVED AIR
TABLE 1
- $9
CULTURES)
10.4 __+0.4 **
11.0 + 0 . 5 **
9.4 +0.4
9.4 _+0.3
+ $9
S t a r v e d air
15.7 + 0 . 7 a***
14.7 + 0 . 6 ***
10.9 +0.5 *
11.5 _+0.4 ***
- $9
BY S M O K E E M I S S I O N S
84
95
105
114
126
0.86
1.05
1.08
1.26
1.29
-+3.3 ***
98 _+0.5
10.3
40
5 × 10 - 5 M
9.8
_+0.6 a***
16.6
.
_+0.7 ***
_+0.4 -
.
9,1
a***
***
_+0.4 -
-
_+0.6
13.2
.
_+0.5
17.1
_+0.5 *** . .
11.8
13.7 _+0.5 *** .
11,2
.
_+0.5 *** .
60
60
60
40
40
25
12.7
.
+ 0 . 6 ** .
.
11.0
.
.
.
.
.
_+5.0 ***
89
_+0.4
8.6
8.8 -+0.4
9.4
-
-
-
-
-
-
-+0.4
-
-
-
-
-
* P < 0.05, ** P < 0.01, *** P < 0.001, 2-tailed t test, c o m p a r e d with c o r r e s p o n d i n g control value. a Toxic effects (cell d e a t h , mitotic delay, c h r o m o s o m e f r a g m e n t a t i o n ) . b Because of toxicity, only 30 cells c o u l d be analysed.
(cyclophosphamide)
Positive control
(acetone)
50
76
0.84
Control
50
53
0.54
25
48
0.53
8.0
29.6
0.368
> 100 ***
10.2 -+0.4
10.8 +0.5
+0.4 *
10.4 _+0.8
10.8 _+0.4
14.1 + 0 . 5 a***
16.4 _+0.6 ***
_+0.5 ***
11.4
13.9
-+0.4
12.9 + 0 . 5 **
11.0
_+0.5
10.1
73 _+8.3 ***
8.8 _+0.4
15.0 4-0.6 a***
8.9 _+0.3
9.4 _+0.4
toxic
74 ,,18 a
ai
*' 1 6 = ®
/
I
18~ •
b
~
.,,6
~4
8 0.4
0.8
1.2
1.6 2.0 2.4 2.8 m g dry wood b u r n e d l m l
6.0 ~
2
6
10
14 18 22 mg dry wood b u r n e d / m l
26
Fig. 1. Induction of SCEs in C H O cells treated with smoke emission samples from a wood stove with (a) starved air combustion or (b) normal air combustion burning birch (circles) or spruce (squares). Experiments were carried out with $9 mix (dark symbols) and without $9 mix (open symbols).
carcinogens may need a longer exposure for their metabolic activation (Takehisa and Wolff, 1978; Nachtman and Wolff, 1982). Moreover, the possibility that the toxicity of the samples had interfered with the activity of the $9 enzymes cannot be excluded. Whereas the addition of $9 mix reduced the toxicity of the samples, it is possible that the enhanced mutagenicity of some samples in the presence of $9 in the Salmonella assay may not represent a true metabolic activation effect but rather an unmasking of the direct mutagenicity in the sample, previously undetected because of high toxicity. Consequently, the lack of an S9-mediated response may have resulted from the experimental conditions and may not accurately reflect the absence or presence of promutagens in the samples. The complexity of the activating and deactivating reactions in mixtures of mutagenic compounds makes it difficult to separate the mutagenicity caused by direct acting compounds from the mutagenicity caused by compounds that do not require $9 microsomal activation. Since the metabolic capacity of CHO cells in activating indirect mutagens is known to be extremely poor (Stetka and Wolff, 1976), the direct mutagenicity observed in all the samples firmly suggests that agents other than PAHs are responsible for the SCE induction. The mutagenicity of emissions from wood combustion has not been widely examined in systems other than the Salmonella assay. Using the Salmonella assay, L6froth (1978) showed that combusion emissions of wood chips did not induce mutagenicity in strain TA1535, indicating that mutagens causing base-pair substitutions were not present in detectable concentrations. The absence of mutagenicity in strain TA1535 and the presence of mutagenicity in the frame-shift detector strains strongly indicated that the mutagenic compounds were mainly polycyclic compounds. The high potency of the smoke emissions to induce SCEs in rodent cells, in addition to the earlier information on their capacity to induce response in the Salmonella/microsome assay, emphasizes the significance of combustion emissions as a source of ambient mutagenicity in the human environment.
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Acknowledgements The study received financial support from the Nordic Council of Ministers, through the MIL-2 project and a grant from the Ministry of Interior Affairs, Government of Finland. We thank our colleague Dr. Kaija Linnainmaa for help and advice with the CHO cell system.
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