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6.4. Chromosomal aberrations induced by extracts of airborne particulate matter from fire-exposed and non fire-exposed building ventilation filters
1088
6.4. CHROMOSOMAL ABERRATIONS INDUCED BY EXTRACTS OF AIRBORNE PARTICULATE MATTER FROM FIRE-EXPOSED AND NON FIRE-EXPOSED BUILDING VENTILATION FILTERS T.S.B. Zwanenburg, Sandoz Ltd., Drug Safety Assessment, Toxicology, CH-4002 Basle, Switzerland MATERIAL AND METHODS Cell culture Chinese hamster V79 cells were cultured r:lt 37°C and 5% CO2 as monolayers in Eagles minimal essential medium (MEM), supplemented with 10% fetal calf serum (Gibco), glutamine (2 mM) and 0.2% sodiumbicarbonate, but without antibiotics. The generation time was about 12 hours. The (;ells were free of mycoplasma infection. During the experiments, penicillin (200 IU/m[~ and streptomycin (200 pg/ml) were present in the medium (so-called complete MEIVl medium).
Rat liver S9-mix The same sg-fraction was used as in the Ames tests (see W. Surer, this issue). The S9-mix was prepared In the following way: I,|ADP (2.24 g/I) and glucose-6-phosphate (G-6-P, 5.07 g/I) were dissolved in distilled water on the day of treatment. Then, per liter, 100 ml Hanks BSS (10 x concentrated), 300 ml NADPIG-6-P solution, 292 ml H20, 7.95 ml NaHCO3 (4.4%), and 300 ml S9-fraction were mixed and stored on ice.
Filter extract dilutions Shortly before application, a filter extract sohJtion sample (120 cm2/ml) was taken and a series of dilutions was made in methanol Then the solutions were transferred to either complete MEM medium or to S9-mix at a final solvent concentration of 3%. These solutions were prepared immediately before treatment.
Toxicity studies Petri dishes of 9 cm were inoculated with 300 cells in 10 ml of complete MEM medium (2 dishes per point, except for the experiments described in Table 2 : 5 dishes per point). After 16 to 19 hrs the medium was replaced by 5 ml of suitable dilutions of the filter extracts in complete MEM medium or in S9-mix and incubated at 37°C and 5% CO2 for another 3 hrs. The dishes were then washed once with PBS and cultured for 6 days in complete MEM medium. They were then stained with 0.3% crystal violet for 15 min, washed with tap water and dried for 1 hr at 60°C. Colonies with more than 50 cells were counted by eye.
Cytogenetic assay 125,000 cells were seeded in 9 cm petri dishes. After about 48 hrs cells were treated with dilutions of the filter extracts in complete MEM medium or in S9-mix for 3 hrs at 37°C and 5% CO2. Four dishes per point were treated. The dishes were then washed once with PBS (5 ml) and incubated with 10 ml of complete MEM medium for another 13 hrs, the last 2 hrs tn the presence of 0.15 pg/ml colcemid. Then the medium in the
1089
dishes was transferred to 15 ml tubes and the cells were washed carefully with 5 ml PBS. The cells were trypsinized with 5 ml 0.025% Trypsine for 5 min and transferred to the corresponding tubes. Hypotonic treatment (7.5 min in prewarmed 1% sodiumcitrate), fixation (in methanol/acetic acid, 3/1, with three changes) and slide preparation (air-drying procedure) were done according to the standard procedures operative in our laboratory. Cyclophosphamide (CP) and ethyl methane sulphonate (EMS) were included as positive controls with and without metabolic activation, respectively. CP was dissolved in S9-mix at 10 or 15 pM, and EMS at 10, 15 or 20 raM. Slides were stained with 2% Giemsa, in a 40 mM Na2HPO4 - 40 mM NaH2PO4 buffer for 10 min. After drying, the slides were mounted in Depex, coded and analyzed for the presence of chromosomal aberrations. 400 metaphases per treatment (100 per petri dish) were analyzed and the following types of chromosomal aberrations were recorded: chromatid breaks, isolocus breaks, chromatid exchanges, dicentrics, ring chromosomes, double minutes. Cells with more then 5 aberrations were defined as multiple aberrants. Chromatid gaps and isolocus gaps were recorded, but not included In the calculation of the number of abnormal cells.
Data evaluation for extracts of location B Dose-effect relationships were established in two or three experiments for each filter extract. The doses were expressed in m 3 air/ml culture medium. From these doseeffect curves the doses which induced 6% abnormal cells (aproximately a doubling of the base-line frequency) were determined and converted to 'relative clastogenic effectiveness' factors by fixing the dose of the most clastogenic filter at 1.0. The same calculation was done at induction levels of 10% and 20% abnormal cells; the three values were then averaged (Table 1). 'Relative toxicity' was determined using the LCso values.
RESULTS Four pairs of filters (one fire-exposed and one control filter exposed shortly after the fire) from different locations, and, in addition, two selected sets of three ambient air-exposed filters were tested for their capacity to induce chromosomal aberrations and/or cytotoxicity in V79 cells.
STUDIES WITH THE FILTERS FROM LOCATION B BDF02 (FIRE-EXPOSED FILTER) AND BCF02, -08, -09, -10 (CONTROL FILTERS) Chromosomal aberration studies Initial experiments showed that, without S9-mix, chromosomal changes were observed only at concentrations which reduced cell survival to 5% or less. The aberrations seen without S9-mix were, therefore, not considered the result of the clastogenicity of the filter extracts, but to reflect the chromosomal expression of severe metabolic changes in dying cells. Upon addition of S9-mix, chromosomal aberrations started to appear at concentrations which reduced cell survival only slightly. Therefore, in the main study, only extracts which had been metabolically activated by an S9-mix were tested All filters from location B induced dose-dependent increases in the frequency of chromosomal aberrations. Only chromatid-type of aberrations were present. Many cells
1090
carrying multiple aberrations were observed at high concentrations. The ranking of clastogenicity in the presence of S9-mix for the various filter extracts was as follows (Table 1): BCF10 < BCF02 < BCF09 < BCF08 < BDF02
Toxicity studies Toxicity in the presence of S9-mix was established in two or three independent experiments. The cytotoxicity ranking (Table 1) was the same as for clastogenicity.
TOXICITY STUDIES WITH FILTERS FROM LOCATIONS A, C, AND D: FILTERS BDF01, -03, -04 (FIRE-EXPOSED FILTERS) AND BCF01, -03, -04, -05, -06, -07 (CONTROL FILTERS) Since there was a very good correlation between the cytotoxicity of the filter extracts and their potential to induce chromosomal aberrations in the V79 cells, as seen in experiments with extracts from location B ('lal:,le 1), the filters from other locations were examined for toxicity only (Table 2). With no exceptions, the toxicity of all the fire.exposed filters was similar to that of the controls exposed shortly after the fire. Similar toxicity was obtained with filters BCF05,06,-07, which were in use in March/April 1987, at location A. For comparison, the toxicity of the filter extracts from location B is included. The filters of this location (with the exception of BCF10) were shown to be much more cytotoxlc than the filters from all other locations.
DISCUSSION Induction of chromosomal aberrations and SC,E's by extracts of ambient airborne particulate matter was previously reported by Alink et al., 1983; Hadnagy et al.; 1986; Krishna et al., 1984; Krishna et al. 1986; de Raat, 1983; Viau et al., 1982. However, since all of these investigations except one (de Raat, 1983) were carried out under non-metabolizing conditions, a comparison of results with the present study is difficult. Results of this study strongly suggest that metabolic activation should be included if extracts of airborne particulate matter are tested for clastogenic activity. As demonstrated by the results of the filter extracts from location B (filters BDF02 and BCF02,-08,-09,-10), there was a good correlation between the ability of a filter extract to induce chromosomal aberrations and cell killing in the V79 cells. Because of this correlation, extracts from the other locations (filters BDF01, -03, -04 and BCF01, -03, -04, -05, -06, -07) were investigated for cytotoxicity only. The following conclusions can be drawn. For locations A, C, and D (filters BDF01BCF01, BCF05,-06,-07; BDF03-BCF03 and BDF04-BCF04, there was no major difference in cytotoxicity between extracts from fire-exposed and control filters, also indicating that no increased clastogenic activity was present in extracts from fireexposed filters. Only one filter, the fire-exposed one from location B (BDF02), contained more (by a factor of about 2) clastogenic material than the control (BCF02) exposed shortly after the fire. The three control filters from the same location (BCF08,-
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09,-10), which were exposed to urban air for three consecutive periods of 10 or 11 days some four months later, displayed large differences in clastogenicity. While the extract from filter BCF10, exposed from March 26 to April 6, 1987, was half as clastogenic as the extract from the fire-exposed filter, the extract from the filter exposed from March 6 to March 16, 1987 (BCF08) was only slightly less clastogenic than the fire-exposed one. In agreement with the Ames test results (W. Suter, this issue), extracts from location B were considerably more (geno)toxic than those from the other locations, whether or not extracts were from fire-exposed filters. As already found in the Ames tests, there were large variations in results between extracts from different locations and among extracts from the same location but collected at different times. In summary, the presented data gave no indication that the fire in Schweizerhalle produced clastogenic material in quantities which would have increased clastogenic activity and/or cytotoxicity of filter extracts substantially above normal city levels (for more details see Zwanenburg, in press).
1093
TABLE
2: Cell
survival with extracts
correspondin~
to 4
and 6 m* air/ml (for this
filter issue)
Code
code
identification
Location
see K.E.
Survival at 4 m' air/ml
Suter
and G r ~ n i n g e r ,
Survival at 6 m J air/ml
% BDF01 BCF01
A A
i00 99
65 65
BCF05 BCF06 BCF07
A A A
103 107 96
73 59 63
BCF03 BCF03
109 102
81 76
BDF04 BCF04
105 106
71 80
BDF02 BCF02
0 85
0 20
BCF08 BCF09 BCFI0
20 50 i00
15 60
4
6.4. CHROMOSOMAL ABERRATIONS INDUCED BY EXTRACTS OF AIRBORNE PARTICULATE MATTER FROM FIRE-EXPOSED AND NON FIRE-EXPOSED BUILDING VENTILATION FILTERS T.S.B. Zwanenburg, Sandoz Ltd., Drug Safety Assessment, Toxicology, CH-4002 Basle, Switzerland MATERIAL AND METHODS Cell culture Chinese hamster V79 cells were cultured r:lt 37°C and 5% CO2 as monolayers in Eagles minimal essential medium (MEM), supplemented with 10% fetal calf serum (Gibco), glutamine (2 mM) and 0.2% sodiumbicarbonate, but without antibiotics. The generation time was about 12 hours. The (;ells were free of mycoplasma infection. During the experiments, penicillin (200 IU/m[~ and streptomycin (200 pg/ml) were present in the medium (so-called complete MEIVl medium).
Rat liver S9-mix The same sg-fraction was used as in the Ames tests (see W. Surer, this issue). The S9-mix was prepared In the following way: I,|ADP (2.24 g/I) and glucose-6-phosphate (G-6-P, 5.07 g/I) were dissolved in distilled water on the day of treatment. Then, per liter, 100 ml Hanks BSS (10 x concentrated), 300 ml NADPIG-6-P solution, 292 ml H20, 7.95 ml NaHCO3 (4.4%), and 300 ml S9-fraction were mixed and stored on ice.
Filter extract dilutions Shortly before application, a filter extract sohJtion sample (120 cm2/ml) was taken and a series of dilutions was made in methanol Then the solutions were transferred to either complete MEM medium or to S9-mix at a final solvent concentration of 3%. These solutions were prepared immediately before treatment.
Toxicity studies Petri dishes of 9 cm were inoculated with 300 cells in 10 ml of complete MEM medium (2 dishes per point, except for the experiments described in Table 2 : 5 dishes per point). After 16 to 19 hrs the medium was replaced by 5 ml of suitable dilutions of the filter extracts in complete MEM medium or in S9-mix and incubated at 37°C and 5% CO2 for another 3 hrs. The dishes were then washed once with PBS and cultured for 6 days in complete MEM medium. They were then stained with 0.3% crystal violet for 15 min, washed with tap water and dried for 1 hr at 60°C. Colonies with more than 50 cells were counted by eye.
Cytogenetic assay 125,000 cells were seeded in 9 cm petri dishes. After about 48 hrs cells were treated with dilutions of the filter extracts in complete MEM medium or in S9-mix for 3 hrs at 37°C and 5% CO2. Four dishes per point were treated. The dishes were then washed once with PBS (5 ml) and incubated with 10 ml of complete MEM medium for another 13 hrs, the last 2 hrs tn the presence of 0.15 pg/ml colcemid. Then the medium in the
1089
dishes was transferred to 15 ml tubes and the cells were washed carefully with 5 ml PBS. The cells were trypsinized with 5 ml 0.025% Trypsine for 5 min and transferred to the corresponding tubes. Hypotonic treatment (7.5 min in prewarmed 1% sodiumcitrate), fixation (in methanol/acetic acid, 3/1, with three changes) and slide preparation (air-drying procedure) were done according to the standard procedures operative in our laboratory. Cyclophosphamide (CP) and ethyl methane sulphonate (EMS) were included as positive controls with and without metabolic activation, respectively. CP was dissolved in S9-mix at 10 or 15 pM, and EMS at 10, 15 or 20 raM. Slides were stained with 2% Giemsa, in a 40 mM Na2HPO4 - 40 mM NaH2PO4 buffer for 10 min. After drying, the slides were mounted in Depex, coded and analyzed for the presence of chromosomal aberrations. 400 metaphases per treatment (100 per petri dish) were analyzed and the following types of chromosomal aberrations were recorded: chromatid breaks, isolocus breaks, chromatid exchanges, dicentrics, ring chromosomes, double minutes. Cells with more then 5 aberrations were defined as multiple aberrants. Chromatid gaps and isolocus gaps were recorded, but not included In the calculation of the number of abnormal cells.
Data evaluation for extracts of location B Dose-effect relationships were established in two or three experiments for each filter extract. The doses were expressed in m 3 air/ml culture medium. From these doseeffect curves the doses which induced 6% abnormal cells (aproximately a doubling of the base-line frequency) were determined and converted to 'relative clastogenic effectiveness' factors by fixing the dose of the most clastogenic filter at 1.0. The same calculation was done at induction levels of 10% and 20% abnormal cells; the three values were then averaged (Table 1). 'Relative toxicity' was determined using the LCso values.
RESULTS Four pairs of filters (one fire-exposed and one control filter exposed shortly after the fire) from different locations, and, in addition, two selected sets of three ambient air-exposed filters were tested for their capacity to induce chromosomal aberrations and/or cytotoxicity in V79 cells.
STUDIES WITH THE FILTERS FROM LOCATION B BDF02 (FIRE-EXPOSED FILTER) AND BCF02, -08, -09, -10 (CONTROL FILTERS) Chromosomal aberration studies Initial experiments showed that, without S9-mix, chromosomal changes were observed only at concentrations which reduced cell survival to 5% or less. The aberrations seen without S9-mix were, therefore, not considered the result of the clastogenicity of the filter extracts, but to reflect the chromosomal expression of severe metabolic changes in dying cells. Upon addition of S9-mix, chromosomal aberrations started to appear at concentrations which reduced cell survival only slightly. Therefore, in the main study, only extracts which had been metabolically activated by an S9-mix were tested All filters from location B induced dose-dependent increases in the frequency of chromosomal aberrations. Only chromatid-type of aberrations were present. Many cells
1090
carrying multiple aberrations were observed at high concentrations. The ranking of clastogenicity in the presence of S9-mix for the various filter extracts was as follows (Table 1): BCF10 < BCF02 < BCF09 < BCF08 < BDF02
Toxicity studies Toxicity in the presence of S9-mix was established in two or three independent experiments. The cytotoxicity ranking (Table 1) was the same as for clastogenicity.
TOXICITY STUDIES WITH FILTERS FROM LOCATIONS A, C, AND D: FILTERS BDF01, -03, -04 (FIRE-EXPOSED FILTERS) AND BCF01, -03, -04, -05, -06, -07 (CONTROL FILTERS) Since there was a very good correlation between the cytotoxicity of the filter extracts and their potential to induce chromosomal aberrations in the V79 cells, as seen in experiments with extracts from location B ('lal:,le 1), the filters from other locations were examined for toxicity only (Table 2). With no exceptions, the toxicity of all the fire.exposed filters was similar to that of the controls exposed shortly after the fire. Similar toxicity was obtained with filters BCF05,06,-07, which were in use in March/April 1987, at location A. For comparison, the toxicity of the filter extracts from location B is included. The filters of this location (with the exception of BCF10) were shown to be much more cytotoxlc than the filters from all other locations.
DISCUSSION Induction of chromosomal aberrations and SC,E's by extracts of ambient airborne particulate matter was previously reported by Alink et al., 1983; Hadnagy et al.; 1986; Krishna et al., 1984; Krishna et al. 1986; de Raat, 1983; Viau et al., 1982. However, since all of these investigations except one (de Raat, 1983) were carried out under non-metabolizing conditions, a comparison of results with the present study is difficult. Results of this study strongly suggest that metabolic activation should be included if extracts of airborne particulate matter are tested for clastogenic activity. As demonstrated by the results of the filter extracts from location B (filters BDF02 and BCF02,-08,-09,-10), there was a good correlation between the ability of a filter extract to induce chromosomal aberrations and cell killing in the V79 cells. Because of this correlation, extracts from the other locations (filters BDF01, -03, -04 and BCF01, -03, -04, -05, -06, -07) were investigated for cytotoxicity only. The following conclusions can be drawn. For locations A, C, and D (filters BDF01BCF01, BCF05,-06,-07; BDF03-BCF03 and BDF04-BCF04, there was no major difference in cytotoxicity between extracts from fire-exposed and control filters, also indicating that no increased clastogenic activity was present in extracts from fireexposed filters. Only one filter, the fire-exposed one from location B (BDF02), contained more (by a factor of about 2) clastogenic material than the control (BCF02) exposed shortly after the fire. The three control filters from the same location (BCF08,-
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09,-10), which were exposed to urban air for three consecutive periods of 10 or 11 days some four months later, displayed large differences in clastogenicity. While the extract from filter BCF10, exposed from March 26 to April 6, 1987, was half as clastogenic as the extract from the fire-exposed filter, the extract from the filter exposed from March 6 to March 16, 1987 (BCF08) was only slightly less clastogenic than the fire-exposed one. In agreement with the Ames test results (W. Suter, this issue), extracts from location B were considerably more (geno)toxic than those from the other locations, whether or not extracts were from fire-exposed filters. As already found in the Ames tests, there were large variations in results between extracts from different locations and among extracts from the same location but collected at different times. In summary, the presented data gave no indication that the fire in Schweizerhalle produced clastogenic material in quantities which would have increased clastogenic activity and/or cytotoxicity of filter extracts substantially above normal city levels (for more details see Zwanenburg, in press).
1093
TABLE
2: Cell
survival with extracts
correspondin~
to 4
and 6 m* air/ml (for this
filter issue)
Code
code
identification
Location
see K.E.
Survival at 4 m' air/ml
Suter
and G r ~ n i n g e r ,
Survival at 6 m J air/ml
% BDF01 BCF01
A A
i00 99
65 65
BCF05 BCF06 BCF07
A A A
103 107 96
73 59 63
BCF03 BCF03
109 102
81 76
BDF04 BCF04
105 106
71 80
BDF02 BCF02
0 85
0 20
BCF08 BCF09 BCFI0
20 50 i00
15 60
4