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Comparative in vivo and in vitro genotoxicity studies of airborne particle extract in mice
Mutation Research, 171 (1986) 157-163
157
Elsevier MTR 01090
Comparative in vivo and in vitro genotoxicity studies of airborne particle extract in mice G. Krishna 1, j. Nath 2, L. Soler 2 and T. Ong 1,2 I National Institute for Occupational Safety and Health, Division of Respiratory Disease Studies, Morgantown, W V 26506-2888 and 2 Division of Plant and Soil Sciences, West Virginia University, Morgantown, W V 26506-6108 (U.S.A.)
(Received9 December1985) (Revisionreceived 3 March 1986) (Accepted 28 March 1986)
Summary The genotoxicity of an acetone extract of locally collected airborne particles was evaluated both in vitro and in vivo using the sister-chromatid exchange (SCE) assay in mice. At the highest concentration (5.36 m g / 5 ml culture), the extract caused approximately a 3-fold increase in SCEs over controls in mouse bone marrow and spleen primary cells in vitro. However, the same airborne particle extract did not induce a significant increase in the SCE level over controls in vivo in mouse bone marrow and spleen cells when administered intraperitoneally or through oral gavage. This indicates that bone marrow and spleen primary cell cultures can be used in in vitro genotoxicity studies of complex mixtures, and that the genotoxicity of airborne particles detected in the in vitro system cannot always be detected in vivo with the same cell types. In addition, the same acetone extract of airborne particles caused dose-related his ÷ revertants in the strain TA98 of S a l m o n e l l a t y p h i m u r i u m , both with and without $9 activation. The significant finding of this study is that the in vitro genotoxicity results of airborne particle extract may not be very meaningful in an in vivo situation.
In recent years many studies have been conducted to assess the carcinogenic risk and genotoxic hazards posed by environmental agents such as air pollutants. The solvent extracts of airborne particles from many locations and occupational settings have been shown to be mutagenic in the Ames Salmonella assay (Chrisp and Fisher, 1980; Epler, 1980; Hughes et al., 1980; Holmberg and Ahlborg, 1983). To date, only a few short-term in vitro genotoxicity studies of airborne particles in mammalian cells have been carried out. Solvent t
Address for correspondence: Tong-man Ong, Ph.D. NIOSH, MicrobiologySection,944 Chestnut Ridge Road, Morgantown, WV 26505-2888(U.S.A.).
extracts of city-smog from the heavily industrialized Rhine-Ruhr area of West-Germany (Schurer et al., 1980; Seemayer et al., 1982), and airborne particles from Lexington, Kentucky (Lockard et al., 1981) were found to induce sister-chromatid exchanges (SCEs) in human peripheral lymphocytes. Airborne particle extracts from non-industrial locations of Wageningen (The Netherlands), have induced SCEs in Chinese hamster lung (V79) cells (Alink et al., 1983). In a previous study, locally collected airborne particle extracts caused dose-related SCEs and chromosomal aberrations in human peripheral lymphocytes (Krishna et al., 1984). Recently, Walker et al. (1982) have correlated
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158 the mutagenicity level of Houston air particulate extracts with lung cancer mortality, as assessed by the Ames Salmonella assay. Extracts of airborne particles from central Tokyo induced mutations in the Ames assay and neutral fractions of these extracts caused a significantly high number of lung tumors and thymic lymphomas in mice over 52 weeks after treatment (Sasaki et al., 1984). Cytogenetic analysis has been used for monitoring the hazards of chemicals to exposed populations. Because of the potential health hazard of air pollution to the exposed population, it would be of interest to determine whether solvent extracts of airborne particles can cause genetic damage/alterations in in vivo situations. Since metabolic activation, detoxification, transportation and distribution of a test compound occur in the intact animal, it would also be of interest to perform in vivo and in vitro comparison studies using the same cell types. The SCE assay has been adapted for both in vivo and in vitro genotoxicity studies in specific target tissues and organs (Tice et al., 1984; Pereira et al., 1982). It has also been demonstrated to be a sensitive measure of DNA damage and carcinogenic potential (Latt et al., 1981). This study was designed to determine whether the solvent extract of airborne particles collected locally could induce SCEs in in vitro and in vivo mouse bone marrow and spleen cells. The same extract was also tested for its genotoxicity in Salmonella typhimurium using the Ames assay. Materials and methods
Sample collection and preparation. Airborne particles were collected, extracted and prepared as described in a previous study (Krishna et al., 1985b). In brief, samples were collected with a Hi-Vol sampler during October 1984 on high-purity glass microfiber filters. The collected samples were soaked in acetone for 30 rain and then the extract was filtered through Whatman No. 2 filter paper. The acetone extract was concentrated to approximately 10 ml with a rotary evaporator (40°C) and then to dryness on a dry bath (40°C) under a stream of nitrogen gas. The dried extracts were dissolved in reagent grade dimethylsulfoxide (DMSO) and were either used immediately or
stored at - 20°C until used. The amount of DMSO in different concentrations of airborne particles tested was the same, i.e., 0.1 ml/plate for the Ames test or 0.1 m l / 5 ml culture medium for the in vitro SCE assay. However, for in vivo studies the extract was first dissolved in a minimal amount of DMSO and then diluted with corn oil. Animals and chemicals. The 6-8 week old CD 1 male mice, weighing 25-30 g, were purchased from Charles River Breeding Laboratories, Inc., Kingston, NJ and raised under normal room temperature and relative humidity conditions. Cyclophosphanlide (Sigma Chemical Company, St. Louis, MO) was used as a positive control for the in vivo SCE assay and trinitrofluorenone (Sigma) was used as a positive control in the in vitro SCE and Ames assays (without $9 activation). 2Aminoanthracene (Sigma) was used as a positive control in the Ames assay with $9 activation.
In vitro sister-chromatid exchange assay Bone marrow removal and culture. The bone marrow culturing procedure has been described in a previous study (Krishna et al., 1985c). In brief, femora and tibia were removed from mice and freed of adherent muscle and cleaned with 70% ethyl alcohol. The tips of bones were then removed with scissors. The marrow was flushed out with Ham's F-12 medium (Flow Laboratories, McLean, VA) into a 15-ml centrifuge tube. Following removal of debris, tubes were centrifuged at 285 × g for 6 rain. The supernatant was removed and the pellet resuspended with the remaining solution. Bone marrow cells (approximately 1.5 million) were incubated in a 25-cm2 Falcon flask with 5 ml of the following complete medium: 3.45 ml Ham's F-12, 1 ml fetal bovine serum (20%) (Gibco), 0.05 ml penicillin-streptomycin (1%) (Gibco), 0.5 ml pregnant mouse uterus extract (10%), and 20 /~M 5-bromo-2'-deoxyuridine (BrdU, Sigma). Cultures were then covered with aluminum foil and incubated at 37°C. 8 h after the start of incubation, negative, positive and test compounds were added. After 30 h of incubation, colchicine (33 /~M final concentration, Gibco) was added and cells were harvested 4 h later. Cell harvest, slide preparation and staining. The contents of the flasks were decanted into 15-ml
159
Falcon centrifuge tubes. The flasks were rinsed with 3 ml Hanks' balanced salt solution which was then transferred to the centrifuge tubes. The tubes were centrifuged at 285 × g for 6 min. The supernatant was removed and the cell pellet resuspended in 5 ml hypotonic solution (0.075 M KC1 at 37 ° C) for 20 rain and recentrifuged. The cells were fixed twice, each time with 5 ml of freshly prepared 3 : 1, methanol : acetic acid fixative. Finally, the cell suspension was diluted with a few drops of fixative a n d dropped on precleaned chilled wet slides which were then air dried for 24 h. Staining for SCE analysis was performed according to a modified technique of Perry and Wolff (1974) and Goto et al. (1978). Slides were stained for 15 rain with Hoechst 33258 (5 ~tg/ml) and exposed to "black" light at 5 5 - 6 0 ° C for 15 rain at a distance of 1 cm while immersed in Sorenson's buffer (phosphate buffer, pH 6.8). The slides were then rinsed with distilled water and stained with 5% Giemsa (in Sorenson's buffer) for i 0 - 1 5 min. All slides were coded and analyzed for SCEs. The replicative index ( R I ) was Calculated as follows: RI= 1M~+2M 2+3M 3 100 where M 1, M 2, and M 3 represent proportions of first-, second-, and third-plus-subsequent-division metaphases respectively (Krishna et al., 1985a). Spleen removal and cell culture. Spleens were obtained aseptically by opening the abdominal cavity of the same mice that were used for bone marrow collection. Each spleen was then transferred into a centrifuge tube containing 2 ml RPMI 1640 with L-glutamine and Hepes-buffered medium (Gibco) supplemented with 20% heat-inactivated fetal bovine serum (Gibco), 2 mM additional L-glutamine (Gibco) and 1% penicillin and streptomycin (Gibco). The spleen was smashed using a sterile spatula. Debris was removed and cells were washed three times with phosphatebuffered saline containing 2% heat-inactivated fetal bovine serum by centrifugation at 285 × g for 6 min. The final cell suspension (approximately 1.5 million cells) was cultured in 5 ml medium consisting of: 3.70 ml RPMI 1640 with
L-glutamine and Hepes buffer, 1.0 ml heat-inactivated fetal bovine serum (20%), 0.05 ml penicillin-streptomycin (1%), 0.05 ml 200 mM Lglutamine solution, 1 × 10 -5 M 2-mercaptoethanol (Sigma), 20 /~M BrdU, and 0.2 ml lipopolysaccharide (Escherichia coil serotype 0111 : B4; Sigma, stock of 600 btg/ml in phosphate-buffered saline). The cell suspension with the complete medium was dispensed into 25-cm2 Falcon tissue-culture flasks, covered with aluminum foil, and then incubated at 37°C with 98% relative humidity and 5% CO 2. After 8 h of incubation, negative, positive and test compounds were added. After 40 h of incubation, colchicine (33/tM final concentration) was added and cells were harvested 4 h later. The harvest, slide preparation, staining and scoring were performed as described for bone marrow cells. In vivo sister-chromatid exchange assay For toxicity studies, 5 different concentrations (60, 179, 536, 1073 and 1609 mg per kg body weight) of airborne particle extract were dosed intraperitoneally as well as through oral gavage. 5 animals were used for each treatment. The two highest concentrations (1073 and 1609 mg per kg body weight) were toxic to the animals and caused death within 24 h. Thus, these concentrations were not included in the follow-up genotoxicity studies. Bone marrow. The positive control chemical (cyclophosphamide, 10 m g / k g body weight), the negative control vehicle (corn oil with 20% DMSO), and the airborne particle extract were injected intraperitoneally (i.p.) as well as by oral gavage into experimental animals (with a volume of approximately 0.3 ml per animal). The paraffin-coated BrdU tablets (50 mg, Boehringer Mannheim Biochemicals, Indianapolis, IN) were inserted under the skin on the flank 1 h after injection of the test compounds (McFee et al.~ 1983). A commercial diet (Purina Certified Laboratory Chow, r a t and mouse blox) and water were given ad libitum until the animals were sacrificed. At 21 h after BrdU tablet implantation, animals were injected with colchicine (4 m g / k g ) and sacrificed by cervical dislocation 3 h later. For bone marrow preparations, both femora were removed non-aseptically and the adherent muscle
160 removed. E a c h f e m o r was c l e a n e d with 70% ethyl alcohol a n d the h e a d cut off with scissors. T h e m a r r o w was flushed out with p h y s i o l o g i c a l saline i n t o a centrifuge tube, followed b y c e n t r i f u g a t i o n at 285 × g for 6 min. Cells for S C E analysis were p r e p a r e d as d e s c r i b e d previously. Spleen. Spleens f r o m a n i m a l s used for b o n e m a r r o w were r e m o v e d b y o p e n i n g the a b d o m i n a l cavity. E a c h spleen was transferred into a 15-ml centrifuge t u b e c o n t a i n i n g 2 m l H a n k s ' b a l a n c e d salt solution (Gibco). T h e spleen was s m a s h e d using a spatula. D e b r i s was r e m o v e d a n d cells were treated with h y p o t o n i c solution, fixed a n d slides p r e p a r e d for S C E analysis as d e s c r i b e d for b o n e m a r r o w cells. Mutagenicity assay. Solvent extracts of airb o r n e particles were also tested for their m u t a genic activity in T A 9 8 of S. typhimurium b y the p l a t e - i n c o r p o r a t i o n test with a n d w i t h o u t $9 m e t a b o l i c a c t i v a t i o n ( A m e s et al., 1975). T h e $9 used was p r e p a r e d f r o m livers of Aroclor-1254t r e a t e d (500 m g / k g b o d y weight) m a l e W i s t a r / Lewis rats. A m i n i m u m of 2 plates were used for each of 3 c o n c e n t r a t i o n s of solvent extract. Histid i n e - i n d e p e n d e n t r e v e r t a n t s were scored following i n c u b a t i o n at 37 ° C for 48 h. E a c h e x p e r i m e n t was r e p e a t e d once a n d each d a t a p o i n t r e p r e s e n t s an average of 4 plates. D e t e r m i n a t i o n o f positive
m u t a g e n i c r e s p o n s e was b a s e d on criteria r e c o m m e n d e d b y A m e s et al. (1975). Statistical analysis. T h e genotoxicity results of a i r b o r n e p a r t i c l e extract using S C E assays, b o t h in vitro a n d in vivo, a n d the A m e s test were c o m p a r e d to negative c o n t r o l values b y S t u d e n t ' s ' t ' test. Also, c o r r e l a t i o n coefficients ( r ) of dose a n d r e s p o n s e were c a l c u l a t e d for in vitro SCE a n d A m e s assays. Results and discussion
T h e in vitro S C E d a t a on m o u s e b o n e m a r r o w a n d spleen cells following t r e a t m e n t with a i r b o r n e p a r t i c l e extract are p r e s e n t e d in T a b l e 1. T h e SCE f r e q u e n c y i n c r e a s e d with increasing doses of a i r b o r n e p a r t i c u l a t e extract. C o m p a r a b l e d o s e responses were o b t a i n e d in b o t h b o n e m a r r o w ( r = 0.97) a n d spleen ( r = 0.96) cells. A t a c o n c e n t r a t i o n of 5.36 mg p e r 5 ml culture, a p p r o x i m a t e l y a 3-fold increase in SCE level over controls was n o t i c e d in b o t h cell types studied. However, at the highest c o n c e n t r a t i o n tested (16.09 m g p e r 5 m l culture), a toxic effect was n o t i c e d in b o t h b o n e m a r r o w a n d spleen cell cultures. These results showed that the solvent extract of locally collected a i r b o r n e particles i n d u c e d SCEs in b o t h b o n e m a r r o w a n d spleen cells in p r i m a r y
TABLE 1 SISTER-CHROMATID EXCHANGES INDUCED BY SOLVENT EXTRACT OF AIRBORNE PARTICLES IN MOUSE PRIMARY BONE MARROW AND SPLEEN CELL CULTURES a Airborne particles (mg per 5 ml culture)
Bone marrow
SCEs/cell __.S.D.
R.I. + S.D.
Spleen SCEs/cell + S.D.
R.I. + S.D.
Negative control b
6.47 + 0.22
1.99 + 0.08
6.55 + 0.24
1.98 -I-0.10
Positive control c
22.05 _+0.55
1.95 + 0.01
21.31 + 0.38
2.01 + 0.19
0.60 1.79 5.36 16.09
8.97 + 0.52 d 13.09 + 0.82 d 18.67 + 0.80 d Only occasional first division metaphases
1.95 + 0.13 1.96 + 0.02 1.79 + 0.08
8.96 + 0.32 d 12.83 + 0.30 d 17.35 + 0.83 d Only occasional first division metaphases observed
1.94 + 0.04 1.90 + 0.04 1.80 + 0.06
observed
a 3 animal cell cultures were used for each treatment and 25 second-division metaphases were scored from each culture. The concentration of airborne particles indicated was based on original particle weight. b 0.1 ml dimethylsulfoxide per 5 ml culture. c 2.0 # g trinitrofluorenone per 5 ml culture. Significant at p < 0.01, over controls.
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TABLE 2 SISTER-CHROMATID E X C H A N G E F R E Q U E N C I E S IN BONE MARROW A N D SPLEEN CELLS A F T E R MICE WERE TREATED W I T H A I R B O R N E PARTICLE EXTRACT BY I N T R A P E R I T O N E A L I N J E C T I O N a Airborne particles
Bone marrow
(mg per kg body weight)
SCEs/ceU ± S.D.
Spleen R.I. ± S.D.
SCEs/cell ± S.D.
R.I. ± S.D.
Negative control b
2.96 + 0.45
1.93 ± 0.06
2.68 ± 0.31
2.03 _+0.10
Positive control c
24.15 + 1.06
1.97 ± 0.10
22.44 ± 0.88
2.04 ± 0.09
2.91 ± 0.13 3.04 ± 0.29 3.05 ± 0.12
1.93 ± 0.07 1.96 ± 0.07 1.96 ± 0.05
2.69 ± 0.22 2.85 + 0.22 2.93 ± 0.13
1.98 ± 0.02 1.86 ± 0.09 1.93 ± 0.12
60 179 536
a 3 animals were used for each treatment and 25 second-division metaphases were scored from each animal. The concentration of airborne particles indicated was based on original particle weight. b -- 0.3 ml solvent vehicle (corn oil with 20% dimethylsulfoxide per animal). c 10 mg cyclophosphamide per kg body weight ( - 0.3 ml per animal).
cell culture. In addition, these cell types are equally sensitive to the genotoxic effects of airborne particles. The induction of SCEs in cell culture in the absence of metabolic activation suggests the presence of direct-acting mutagens in the airborne particle extract studied. These results are in agreement with other reports (Krishna et al., 1984; Lockard et al., 1981; Alink et al., 1983). Compounds responsible for mutagenic activity of airborne particles studied are not known. However, it is surmised that common mutagenic/clastogenic components of solvent extracts of airborne particles may be derivatives of polycyclic aromatic hydrocarbons (Pitts et al., 1977; Talcott and We±, 1977; Wang et al., 1978; Whong et al., 1981).
The frequencies of SCEs in vivo after i.p. injection and oral gavage of different concentrations of airborne particle extract are shown in Tables 2 and 3, respectively. Airborne particle extract, with different dosages, did not induce significant increase in SCE levels over controls in either bone marrow or spleen cells. This observation was consistent in both routes of administration of the extract. Because of the toxicity, concentrations higher than those used in this study, were not tested. The negative results of airborne particles in the in vivo SCE assay may be due to: the inactivation of mutagenic compounds in the body, the inability of mutagenic compounds to reach the bone marrow and spleen or the relative low con-
TABLE 3 SISTER-CHROMATID E X C H A N G E FREQUENCIES IN BONE MARROW A N D SPLEEN CELLS A F T E R MICE WERE TREATED W I T H A I R B O R N E PARTICLE EXTRACT BY ORAL GAVAGE a Airborne particles
Bone marrow
(mg per kg body weight)
SCEs/cell ± S.D.
Spleen R.I. ± S.D.
SCEs/cell + S.D.
R.I. ± S.D.
Negative control b
2.76 + 0.08
1.90 + 0.04
2.55 + 0.06
2.03 + 0.09
Positive control ¢
21.95 + 0.75
1.96 ± 0.14
19.48 + 0.85
1.94 + 0.05
2.81 + 0.35 2.60 + 0.24 2.79 + 0.22
1.97 + 0.06 2.00 + 0.08 1.97 + 0.07
2.75 + 0.09 2.85 + 0.24 2.76 + 0.36
1.95 + 0.05 1.87 _+0.11 1.94 + 0.03
60 179 536
a 3 animals were used for each treatment and 25 second-division metaphascs were scored from each animal. The concentration of airborne particles indicated was based on original particle weight. b -- 0.3 ml solvent vehicle (corn oil with 20% dimethylsulfoxide per animal). c 10 nag cyclophosphamide per kg body weight ( - 0.3 ml per animal).
162
centration of mutagenic material in these organs to cause significant increase in SCE level. It is also possible that the airborne particle extract is metabolized before it reaches the target cells. The positive control compound, cyclophosphamide (10 m g / k g body weight), caused a dramatic increase in SCE level under these conditions. The in vivo induction of SCEs in bone marrow and spleen cells following cyclophosphamide treatment is in agreement with earlier studies (Allen et al., 1977; Latt et al., 1981). The results of the airborne particle extract reported here are similar to some of the chemical compounds such as proflavine, methylene blue, chloropromazine (Speit, 1982), and ethylene dibromide (Krishna et al., 1985a), which are known to induce SCEs in vitro; however, they did not induce significant dose-related SCEs in vivo in mammalian systems. It has been reported that the neutral fraction of airborne particle extract causes slight but significant increase in numbers of lung tumors and thymic lymphomas in mice (Sasaki et al., 1984). In the present study, whole extract was used which may not be as genotoxic as the neutral fraction. In addition, the duration of treatment may be important for potential genotoxicity of airborne particles in in vivo situations. It may be observed that the amount and type of mutagens in airborne particles may vary with environmental and meteorological conditions. To properly evaluate the potential genotoxicity and carcinogenicity of air particulate extract in vivo, chronic studies may have to be conducted. Also, other tissues, such as respiratory, as well as other genetic end-points, might be considered for future studies. Since the airborne particle extract caused doserelated SCEs in primary mouse bone marrow and spleen cell cultures, these systems can be used in genotoxicity studies of ambient airborne particles and other environmental complex mixtures. Also, these assays are potentially useful for in vivo and in vitro comparative cytogenetic studies and hence for evaluating the relative value of in vitro assays using the same cell types. In vitro assays assess genotoxic potential while in vivo assays assess the ability of the test compound to express this potential in the organ being tested in an intact animal (Ashby, 1983). The bone marrow and spleen cell data on airborne particles presented here support
the viewpoint that in vitro results are not always predictive of activity in vivo. The positive mutagenic response of airborne particulate extract with the Ames assay (Table 4) is in agreement with our previous reports (Krishna et al., 1984, 1985b). At a concentration of 5.36 mg airborne particulate extract per plate, the average h i s + revertants with and without $9 activation were 563 and 345, respectively. Comparable doserelated mutagenicity was noticed both in the presence ( r = 0.99) and in the absence (r = 0.95) of metabolic activation. A lower frequency of h i s + revertants was observed in the absence (about 8-fold over controls) than in the presence of $9 activation (about ll-fold over controls). However, the highest concentration of airborne particulate extract tested (16.09 mg per plate) yielded lower h i s + revertants in the Ames TA98 strain both with and without $9 activation indicating toxicity at this concentration. It may be noted that all concentrations of airborne particles that were positive in the Ames assay were also positive in the in vitro SCE assay with mouse bone marrow and spleen cells. Recently, good correlations between dose-response of h i s + revertants in Ames test and frequency of
TABLE 4 G E N E MUTATIONS I N D U C E D BY SOLVENT EXTRACT OF A I R B O R N E PARTICLES IN Salmonella typhimurium U S I N G AMES ASSAY a Airborne particles TA98 his + revertants per plate b -1-S.D. (mg per plate) Negative control c Positive control d 0.60 1.79 5.36 16.09 a
b c d e f
With $9 activation Without $9 activation 49.75 + 4.86
39.25 + 2.22
1584.00+80.96
514.50+33.83
115.00+17.42 242.25 + 30.31 563.50 + 38.02 451.50+ 55.31
e e ~ e,f
105.50+17.82 222.75 + 20.71 345.50 + 24.77 233.25 + 20.55
e ~ e e,r
The concentration of airborne particles indicated was based on original particle weight. Average of 2 Expts. with duplicate plates in each experiment. 0.1 ml dimethylsulfoxide per plate. 0.25 /zg 2-aminoanthracene (with $9 activation) and 0.1 #g trinitrofluorenone (without $9 activation) per plate. Significant at p < 0.01, over controls. Toxic to the S. typhimurium.
163 S C E s in C h i n e s e h a m s t e r l u n g (V79) cells a n d human lymphocytes after treatment with airborne particle extract have been demonstrated (Lockard et al., 1981; A l i n k et al., 1983; K r i s h n a et al., 1984). H i g h e r his + r e v e r t a n t s in t h e A m e s a s s a y w i t h $9 a c t i v a t i o n suggests t h e p r e s e n c e of promutagens requiring metabolic activation.
References Alink, G.M., H.A. Smit, J.J. van Houdt, J.R. Kolkman and J.S.M. Boleij (1983) Mutagenic activity of airborne particulates at non-industrial locations, Mutation Res., 116, 21-34. Allen, J.W., C.F. Shuler, R.W. Mendes and S.A. Latt (1977) A simplified technique for vivo analysis of sister chromatid exchanges using 5-bromodeoxyuridine tablets, Cytogenet. Cell Genet., 18, 231-237. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test, Mutation Res., 31, 347-364. Ashby, J. (1983) The unique role of rodents in the detection of possible human carcinogens and mutagens, Mutation Res., 115, 177-213. Chrisp, C.E., and G.L. Fisher (1980) Mutagenicity of airborne particles, Mutation Res., 76, 143-164. Epler, J.L. (1980) The use of short-term tests in the isolation and identification of chemical mutagens in complex mixtures, in: F.J. de Serres and A. Hollaender (Eds.), Chemical Mutagens: Principles and Methods for Their Detection, Vol. 6, Plenum, New York, pp. 239-270. Goto, K., S. Maeda, Y. Kano and T. Sugiyama (1978) Factors involved in differential Giemsa-staining of sister chromatids, Chromosoma, 66, 351-359. Holmherg, B., and U. Ahlborg (1983) Consensus report: Mutagenicity and carcinogenicity of car exhausts and coal combustion emissions, Environ. Health Persp., 47, 1-30. Hughes, T.J., E. Pellizzari, L. Little, C. Sparacino and A. Kolber (1980) Ambient air pollutants: Collection, chemical characterization and mutagenicity testing, Mutation Res., 76, 51-83. Krishna, G., J. Nath and T. Ong (1984) Correlative genotoxicity studies of airborne particles in Salmonella typhimurium and cultured human lymphocytes, Environ. Mutagen., 6, 485-492. Krishna, G., J. Xu, J. Nath, M. Petersen and T. Ong (1985a), In vivo cytogenetic studies on mice exposed to ethylene dibromide, Mutation Res., 158, 81-87. Krishna, G., J. Nath, T. Ong and W.-Z. Whong (1985b) A simple method for the extraction of mutagens from airborne particles, Environ. Monitor. Assessment, 5, 393-398. Krishna, G., J. Nath, T. Ong (1985c) PreparatiOn of mouse bone marrow primary cultures for sister chromatid exchange and chromosomal aberration studies, J. Tissue Culture Methods, 9, 193-198.
Latt, S., J. Allen, S. Bloom, A. Carrano, E. Flak, D. Kram, E. Schneider, R. Schreck, R. Tice, B. Whitfield and S. Wolff (1981) Sister-chromatid exchanges: A report of the GeneTox Program, Mutation Res., 87, 17-62. Lockard, J.M., C.J. Viau, C. Lee-Stephens, J.C. Caldwell, J.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. McFee, A.F., K.W. Lowe and J.R. San Sebastian (1983) Improved sister-chromatid exchange differentiation using paraffin-coated bromodeoxyuridine tablets in mice, Mutation Res., 119, 83-88. Pereira, M.A., L. McMillan, P. Kaur, D.K. Gulati and P.S. Sabharwal (1982) Effect of diesel exhaust emissions, particulates, and exhaust on sister chromatid exchange in transplacentally exposed fetal hamster liver, Environ. Mutagen., 4, 215-220. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature (London), 251, 156-158. Pitts, J.N., D. Grosjean, T.M. Mischke, V.F. Simmon and D. Poole (1977) Mutagenic activity of airborne particulate organic pollutants, Toxicot. Lett., 1, 65-70. Sasaki, Y., R. Endo, T. Kawai, K. Oyama, A. Nakama, T. Ishiguro, K. Suga and K. Asakuno (1984) Mutagenicity and carcinogenicity of extracts from airborne particles, Mutation Res., 130, 379 (Abstract). Schurer, C.C., N. Manojlovic and N.H. Seemayer (1980) Induction of sister chromatid exchange in human cells in vitro by the mutagenic effect of city-smog extract, Mutation Res., 74, 164-165 (Abstract). Seemayer, N.H., N. Manojlovic, C.C. Schurer and H. Behrendt (1982) Application of tissue culture cells in detection of cytotoxic mutagenic and carcinogenic effects of city smog extracts, Second Int. Workshop on the Application of Tissue Cult. in Toxicol., Stockholm, p. 62 (Abstract). Speit, G. (1982) Intercalating substances do not induce sister-chromatid exchanges (SCEs) in vivo, Mutation Res., 104, 261-266. Talcott, R., and E. Wei (1977) Airborne mutagens bioassayed in Salmonella typhimurium, J. Natl. Cancer Inst., 58, 449-451. Tice, R., B. Lambert, K. Morimoto and A. Hollaender (1984) A review of the international symposium on sister chromatid exchanges: Twenty-five years of experimental research, Environ. Mutagen., 6, 737-752. Walker, R.D., T.H. Conner, E.J. MacDonald, N.M. Trieff, M.S. Legator, K.W. MacKenzie and J.G. Dobbins (1982) Correlation of mutagenic assessment of Houston air particulate extracts in relation to lung cancer mortality rates, Environ. Res., 28, 303-312. Wang, Y.Y., S.M. Rappaport, R.F. Sawyer and E.T. Wei (1978) Direct-acting mutagens in automobile exhaust, Cancer Lett., 5, 39-47. Whong, W.-Z., J. Stewart, M. McCawley, P. Major, J.A. Merchant and T. Ong (1981) Mutagenicity of airborne particles from a non-industrial town, Environ. Mutagen., 3, 617-626.
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Elsevier MTR 01090
Comparative in vivo and in vitro genotoxicity studies of airborne particle extract in mice G. Krishna 1, j. Nath 2, L. Soler 2 and T. Ong 1,2 I National Institute for Occupational Safety and Health, Division of Respiratory Disease Studies, Morgantown, W V 26506-2888 and 2 Division of Plant and Soil Sciences, West Virginia University, Morgantown, W V 26506-6108 (U.S.A.)
(Received9 December1985) (Revisionreceived 3 March 1986) (Accepted 28 March 1986)
Summary The genotoxicity of an acetone extract of locally collected airborne particles was evaluated both in vitro and in vivo using the sister-chromatid exchange (SCE) assay in mice. At the highest concentration (5.36 m g / 5 ml culture), the extract caused approximately a 3-fold increase in SCEs over controls in mouse bone marrow and spleen primary cells in vitro. However, the same airborne particle extract did not induce a significant increase in the SCE level over controls in vivo in mouse bone marrow and spleen cells when administered intraperitoneally or through oral gavage. This indicates that bone marrow and spleen primary cell cultures can be used in in vitro genotoxicity studies of complex mixtures, and that the genotoxicity of airborne particles detected in the in vitro system cannot always be detected in vivo with the same cell types. In addition, the same acetone extract of airborne particles caused dose-related his ÷ revertants in the strain TA98 of S a l m o n e l l a t y p h i m u r i u m , both with and without $9 activation. The significant finding of this study is that the in vitro genotoxicity results of airborne particle extract may not be very meaningful in an in vivo situation.
In recent years many studies have been conducted to assess the carcinogenic risk and genotoxic hazards posed by environmental agents such as air pollutants. The solvent extracts of airborne particles from many locations and occupational settings have been shown to be mutagenic in the Ames Salmonella assay (Chrisp and Fisher, 1980; Epler, 1980; Hughes et al., 1980; Holmberg and Ahlborg, 1983). To date, only a few short-term in vitro genotoxicity studies of airborne particles in mammalian cells have been carried out. Solvent t
Address for correspondence: Tong-man Ong, Ph.D. NIOSH, MicrobiologySection,944 Chestnut Ridge Road, Morgantown, WV 26505-2888(U.S.A.).
extracts of city-smog from the heavily industrialized Rhine-Ruhr area of West-Germany (Schurer et al., 1980; Seemayer et al., 1982), and airborne particles from Lexington, Kentucky (Lockard et al., 1981) were found to induce sister-chromatid exchanges (SCEs) in human peripheral lymphocytes. Airborne particle extracts from non-industrial locations of Wageningen (The Netherlands), have induced SCEs in Chinese hamster lung (V79) cells (Alink et al., 1983). In a previous study, locally collected airborne particle extracts caused dose-related SCEs and chromosomal aberrations in human peripheral lymphocytes (Krishna et al., 1984). Recently, Walker et al. (1982) have correlated
0165-1218/86/$03.50 © 1986 ElsevierSciencePublishers B.V. (BiomedicalDivision)
158 the mutagenicity level of Houston air particulate extracts with lung cancer mortality, as assessed by the Ames Salmonella assay. Extracts of airborne particles from central Tokyo induced mutations in the Ames assay and neutral fractions of these extracts caused a significantly high number of lung tumors and thymic lymphomas in mice over 52 weeks after treatment (Sasaki et al., 1984). Cytogenetic analysis has been used for monitoring the hazards of chemicals to exposed populations. Because of the potential health hazard of air pollution to the exposed population, it would be of interest to determine whether solvent extracts of airborne particles can cause genetic damage/alterations in in vivo situations. Since metabolic activation, detoxification, transportation and distribution of a test compound occur in the intact animal, it would also be of interest to perform in vivo and in vitro comparison studies using the same cell types. The SCE assay has been adapted for both in vivo and in vitro genotoxicity studies in specific target tissues and organs (Tice et al., 1984; Pereira et al., 1982). It has also been demonstrated to be a sensitive measure of DNA damage and carcinogenic potential (Latt et al., 1981). This study was designed to determine whether the solvent extract of airborne particles collected locally could induce SCEs in in vitro and in vivo mouse bone marrow and spleen cells. The same extract was also tested for its genotoxicity in Salmonella typhimurium using the Ames assay. Materials and methods
Sample collection and preparation. Airborne particles were collected, extracted and prepared as described in a previous study (Krishna et al., 1985b). In brief, samples were collected with a Hi-Vol sampler during October 1984 on high-purity glass microfiber filters. The collected samples were soaked in acetone for 30 rain and then the extract was filtered through Whatman No. 2 filter paper. The acetone extract was concentrated to approximately 10 ml with a rotary evaporator (40°C) and then to dryness on a dry bath (40°C) under a stream of nitrogen gas. The dried extracts were dissolved in reagent grade dimethylsulfoxide (DMSO) and were either used immediately or
stored at - 20°C until used. The amount of DMSO in different concentrations of airborne particles tested was the same, i.e., 0.1 ml/plate for the Ames test or 0.1 m l / 5 ml culture medium for the in vitro SCE assay. However, for in vivo studies the extract was first dissolved in a minimal amount of DMSO and then diluted with corn oil. Animals and chemicals. The 6-8 week old CD 1 male mice, weighing 25-30 g, were purchased from Charles River Breeding Laboratories, Inc., Kingston, NJ and raised under normal room temperature and relative humidity conditions. Cyclophosphanlide (Sigma Chemical Company, St. Louis, MO) was used as a positive control for the in vivo SCE assay and trinitrofluorenone (Sigma) was used as a positive control in the in vitro SCE and Ames assays (without $9 activation). 2Aminoanthracene (Sigma) was used as a positive control in the Ames assay with $9 activation.
In vitro sister-chromatid exchange assay Bone marrow removal and culture. The bone marrow culturing procedure has been described in a previous study (Krishna et al., 1985c). In brief, femora and tibia were removed from mice and freed of adherent muscle and cleaned with 70% ethyl alcohol. The tips of bones were then removed with scissors. The marrow was flushed out with Ham's F-12 medium (Flow Laboratories, McLean, VA) into a 15-ml centrifuge tube. Following removal of debris, tubes were centrifuged at 285 × g for 6 rain. The supernatant was removed and the pellet resuspended with the remaining solution. Bone marrow cells (approximately 1.5 million) were incubated in a 25-cm2 Falcon flask with 5 ml of the following complete medium: 3.45 ml Ham's F-12, 1 ml fetal bovine serum (20%) (Gibco), 0.05 ml penicillin-streptomycin (1%) (Gibco), 0.5 ml pregnant mouse uterus extract (10%), and 20 /~M 5-bromo-2'-deoxyuridine (BrdU, Sigma). Cultures were then covered with aluminum foil and incubated at 37°C. 8 h after the start of incubation, negative, positive and test compounds were added. After 30 h of incubation, colchicine (33 /~M final concentration, Gibco) was added and cells were harvested 4 h later. Cell harvest, slide preparation and staining. The contents of the flasks were decanted into 15-ml
159
Falcon centrifuge tubes. The flasks were rinsed with 3 ml Hanks' balanced salt solution which was then transferred to the centrifuge tubes. The tubes were centrifuged at 285 × g for 6 min. The supernatant was removed and the cell pellet resuspended in 5 ml hypotonic solution (0.075 M KC1 at 37 ° C) for 20 rain and recentrifuged. The cells were fixed twice, each time with 5 ml of freshly prepared 3 : 1, methanol : acetic acid fixative. Finally, the cell suspension was diluted with a few drops of fixative a n d dropped on precleaned chilled wet slides which were then air dried for 24 h. Staining for SCE analysis was performed according to a modified technique of Perry and Wolff (1974) and Goto et al. (1978). Slides were stained for 15 rain with Hoechst 33258 (5 ~tg/ml) and exposed to "black" light at 5 5 - 6 0 ° C for 15 rain at a distance of 1 cm while immersed in Sorenson's buffer (phosphate buffer, pH 6.8). The slides were then rinsed with distilled water and stained with 5% Giemsa (in Sorenson's buffer) for i 0 - 1 5 min. All slides were coded and analyzed for SCEs. The replicative index ( R I ) was Calculated as follows: RI= 1M~+2M 2+3M 3 100 where M 1, M 2, and M 3 represent proportions of first-, second-, and third-plus-subsequent-division metaphases respectively (Krishna et al., 1985a). Spleen removal and cell culture. Spleens were obtained aseptically by opening the abdominal cavity of the same mice that were used for bone marrow collection. Each spleen was then transferred into a centrifuge tube containing 2 ml RPMI 1640 with L-glutamine and Hepes-buffered medium (Gibco) supplemented with 20% heat-inactivated fetal bovine serum (Gibco), 2 mM additional L-glutamine (Gibco) and 1% penicillin and streptomycin (Gibco). The spleen was smashed using a sterile spatula. Debris was removed and cells were washed three times with phosphatebuffered saline containing 2% heat-inactivated fetal bovine serum by centrifugation at 285 × g for 6 min. The final cell suspension (approximately 1.5 million cells) was cultured in 5 ml medium consisting of: 3.70 ml RPMI 1640 with
L-glutamine and Hepes buffer, 1.0 ml heat-inactivated fetal bovine serum (20%), 0.05 ml penicillin-streptomycin (1%), 0.05 ml 200 mM Lglutamine solution, 1 × 10 -5 M 2-mercaptoethanol (Sigma), 20 /~M BrdU, and 0.2 ml lipopolysaccharide (Escherichia coil serotype 0111 : B4; Sigma, stock of 600 btg/ml in phosphate-buffered saline). The cell suspension with the complete medium was dispensed into 25-cm2 Falcon tissue-culture flasks, covered with aluminum foil, and then incubated at 37°C with 98% relative humidity and 5% CO 2. After 8 h of incubation, negative, positive and test compounds were added. After 40 h of incubation, colchicine (33/tM final concentration) was added and cells were harvested 4 h later. The harvest, slide preparation, staining and scoring were performed as described for bone marrow cells. In vivo sister-chromatid exchange assay For toxicity studies, 5 different concentrations (60, 179, 536, 1073 and 1609 mg per kg body weight) of airborne particle extract were dosed intraperitoneally as well as through oral gavage. 5 animals were used for each treatment. The two highest concentrations (1073 and 1609 mg per kg body weight) were toxic to the animals and caused death within 24 h. Thus, these concentrations were not included in the follow-up genotoxicity studies. Bone marrow. The positive control chemical (cyclophosphamide, 10 m g / k g body weight), the negative control vehicle (corn oil with 20% DMSO), and the airborne particle extract were injected intraperitoneally (i.p.) as well as by oral gavage into experimental animals (with a volume of approximately 0.3 ml per animal). The paraffin-coated BrdU tablets (50 mg, Boehringer Mannheim Biochemicals, Indianapolis, IN) were inserted under the skin on the flank 1 h after injection of the test compounds (McFee et al.~ 1983). A commercial diet (Purina Certified Laboratory Chow, r a t and mouse blox) and water were given ad libitum until the animals were sacrificed. At 21 h after BrdU tablet implantation, animals were injected with colchicine (4 m g / k g ) and sacrificed by cervical dislocation 3 h later. For bone marrow preparations, both femora were removed non-aseptically and the adherent muscle
160 removed. E a c h f e m o r was c l e a n e d with 70% ethyl alcohol a n d the h e a d cut off with scissors. T h e m a r r o w was flushed out with p h y s i o l o g i c a l saline i n t o a centrifuge tube, followed b y c e n t r i f u g a t i o n at 285 × g for 6 min. Cells for S C E analysis were p r e p a r e d as d e s c r i b e d previously. Spleen. Spleens f r o m a n i m a l s used for b o n e m a r r o w were r e m o v e d b y o p e n i n g the a b d o m i n a l cavity. E a c h spleen was transferred into a 15-ml centrifuge t u b e c o n t a i n i n g 2 m l H a n k s ' b a l a n c e d salt solution (Gibco). T h e spleen was s m a s h e d using a spatula. D e b r i s was r e m o v e d a n d cells were treated with h y p o t o n i c solution, fixed a n d slides p r e p a r e d for S C E analysis as d e s c r i b e d for b o n e m a r r o w cells. Mutagenicity assay. Solvent extracts of airb o r n e particles were also tested for their m u t a genic activity in T A 9 8 of S. typhimurium b y the p l a t e - i n c o r p o r a t i o n test with a n d w i t h o u t $9 m e t a b o l i c a c t i v a t i o n ( A m e s et al., 1975). T h e $9 used was p r e p a r e d f r o m livers of Aroclor-1254t r e a t e d (500 m g / k g b o d y weight) m a l e W i s t a r / Lewis rats. A m i n i m u m of 2 plates were used for each of 3 c o n c e n t r a t i o n s of solvent extract. Histid i n e - i n d e p e n d e n t r e v e r t a n t s were scored following i n c u b a t i o n at 37 ° C for 48 h. E a c h e x p e r i m e n t was r e p e a t e d once a n d each d a t a p o i n t r e p r e s e n t s an average of 4 plates. D e t e r m i n a t i o n o f positive
m u t a g e n i c r e s p o n s e was b a s e d on criteria r e c o m m e n d e d b y A m e s et al. (1975). Statistical analysis. T h e genotoxicity results of a i r b o r n e p a r t i c l e extract using S C E assays, b o t h in vitro a n d in vivo, a n d the A m e s test were c o m p a r e d to negative c o n t r o l values b y S t u d e n t ' s ' t ' test. Also, c o r r e l a t i o n coefficients ( r ) of dose a n d r e s p o n s e were c a l c u l a t e d for in vitro SCE a n d A m e s assays. Results and discussion
T h e in vitro S C E d a t a on m o u s e b o n e m a r r o w a n d spleen cells following t r e a t m e n t with a i r b o r n e p a r t i c l e extract are p r e s e n t e d in T a b l e 1. T h e SCE f r e q u e n c y i n c r e a s e d with increasing doses of a i r b o r n e p a r t i c u l a t e extract. C o m p a r a b l e d o s e responses were o b t a i n e d in b o t h b o n e m a r r o w ( r = 0.97) a n d spleen ( r = 0.96) cells. A t a c o n c e n t r a t i o n of 5.36 mg p e r 5 ml culture, a p p r o x i m a t e l y a 3-fold increase in SCE level over controls was n o t i c e d in b o t h cell types studied. However, at the highest c o n c e n t r a t i o n tested (16.09 m g p e r 5 m l culture), a toxic effect was n o t i c e d in b o t h b o n e m a r r o w a n d spleen cell cultures. These results showed that the solvent extract of locally collected a i r b o r n e particles i n d u c e d SCEs in b o t h b o n e m a r r o w a n d spleen cells in p r i m a r y
TABLE 1 SISTER-CHROMATID EXCHANGES INDUCED BY SOLVENT EXTRACT OF AIRBORNE PARTICLES IN MOUSE PRIMARY BONE MARROW AND SPLEEN CELL CULTURES a Airborne particles (mg per 5 ml culture)
Bone marrow
SCEs/cell __.S.D.
R.I. + S.D.
Spleen SCEs/cell + S.D.
R.I. + S.D.
Negative control b
6.47 + 0.22
1.99 + 0.08
6.55 + 0.24
1.98 -I-0.10
Positive control c
22.05 _+0.55
1.95 + 0.01
21.31 + 0.38
2.01 + 0.19
0.60 1.79 5.36 16.09
8.97 + 0.52 d 13.09 + 0.82 d 18.67 + 0.80 d Only occasional first division metaphases
1.95 + 0.13 1.96 + 0.02 1.79 + 0.08
8.96 + 0.32 d 12.83 + 0.30 d 17.35 + 0.83 d Only occasional first division metaphases observed
1.94 + 0.04 1.90 + 0.04 1.80 + 0.06
observed
a 3 animal cell cultures were used for each treatment and 25 second-division metaphases were scored from each culture. The concentration of airborne particles indicated was based on original particle weight. b 0.1 ml dimethylsulfoxide per 5 ml culture. c 2.0 # g trinitrofluorenone per 5 ml culture. Significant at p < 0.01, over controls.
161
TABLE 2 SISTER-CHROMATID E X C H A N G E F R E Q U E N C I E S IN BONE MARROW A N D SPLEEN CELLS A F T E R MICE WERE TREATED W I T H A I R B O R N E PARTICLE EXTRACT BY I N T R A P E R I T O N E A L I N J E C T I O N a Airborne particles
Bone marrow
(mg per kg body weight)
SCEs/ceU ± S.D.
Spleen R.I. ± S.D.
SCEs/cell ± S.D.
R.I. ± S.D.
Negative control b
2.96 + 0.45
1.93 ± 0.06
2.68 ± 0.31
2.03 _+0.10
Positive control c
24.15 + 1.06
1.97 ± 0.10
22.44 ± 0.88
2.04 ± 0.09
2.91 ± 0.13 3.04 ± 0.29 3.05 ± 0.12
1.93 ± 0.07 1.96 ± 0.07 1.96 ± 0.05
2.69 ± 0.22 2.85 + 0.22 2.93 ± 0.13
1.98 ± 0.02 1.86 ± 0.09 1.93 ± 0.12
60 179 536
a 3 animals were used for each treatment and 25 second-division metaphases were scored from each animal. The concentration of airborne particles indicated was based on original particle weight. b -- 0.3 ml solvent vehicle (corn oil with 20% dimethylsulfoxide per animal). c 10 mg cyclophosphamide per kg body weight ( - 0.3 ml per animal).
cell culture. In addition, these cell types are equally sensitive to the genotoxic effects of airborne particles. The induction of SCEs in cell culture in the absence of metabolic activation suggests the presence of direct-acting mutagens in the airborne particle extract studied. These results are in agreement with other reports (Krishna et al., 1984; Lockard et al., 1981; Alink et al., 1983). Compounds responsible for mutagenic activity of airborne particles studied are not known. However, it is surmised that common mutagenic/clastogenic components of solvent extracts of airborne particles may be derivatives of polycyclic aromatic hydrocarbons (Pitts et al., 1977; Talcott and We±, 1977; Wang et al., 1978; Whong et al., 1981).
The frequencies of SCEs in vivo after i.p. injection and oral gavage of different concentrations of airborne particle extract are shown in Tables 2 and 3, respectively. Airborne particle extract, with different dosages, did not induce significant increase in SCE levels over controls in either bone marrow or spleen cells. This observation was consistent in both routes of administration of the extract. Because of the toxicity, concentrations higher than those used in this study, were not tested. The negative results of airborne particles in the in vivo SCE assay may be due to: the inactivation of mutagenic compounds in the body, the inability of mutagenic compounds to reach the bone marrow and spleen or the relative low con-
TABLE 3 SISTER-CHROMATID E X C H A N G E FREQUENCIES IN BONE MARROW A N D SPLEEN CELLS A F T E R MICE WERE TREATED W I T H A I R B O R N E PARTICLE EXTRACT BY ORAL GAVAGE a Airborne particles
Bone marrow
(mg per kg body weight)
SCEs/cell ± S.D.
Spleen R.I. ± S.D.
SCEs/cell + S.D.
R.I. ± S.D.
Negative control b
2.76 + 0.08
1.90 + 0.04
2.55 + 0.06
2.03 + 0.09
Positive control ¢
21.95 + 0.75
1.96 ± 0.14
19.48 + 0.85
1.94 + 0.05
2.81 + 0.35 2.60 + 0.24 2.79 + 0.22
1.97 + 0.06 2.00 + 0.08 1.97 + 0.07
2.75 + 0.09 2.85 + 0.24 2.76 + 0.36
1.95 + 0.05 1.87 _+0.11 1.94 + 0.03
60 179 536
a 3 animals were used for each treatment and 25 second-division metaphascs were scored from each animal. The concentration of airborne particles indicated was based on original particle weight. b -- 0.3 ml solvent vehicle (corn oil with 20% dimethylsulfoxide per animal). c 10 nag cyclophosphamide per kg body weight ( - 0.3 ml per animal).
162
centration of mutagenic material in these organs to cause significant increase in SCE level. It is also possible that the airborne particle extract is metabolized before it reaches the target cells. The positive control compound, cyclophosphamide (10 m g / k g body weight), caused a dramatic increase in SCE level under these conditions. The in vivo induction of SCEs in bone marrow and spleen cells following cyclophosphamide treatment is in agreement with earlier studies (Allen et al., 1977; Latt et al., 1981). The results of the airborne particle extract reported here are similar to some of the chemical compounds such as proflavine, methylene blue, chloropromazine (Speit, 1982), and ethylene dibromide (Krishna et al., 1985a), which are known to induce SCEs in vitro; however, they did not induce significant dose-related SCEs in vivo in mammalian systems. It has been reported that the neutral fraction of airborne particle extract causes slight but significant increase in numbers of lung tumors and thymic lymphomas in mice (Sasaki et al., 1984). In the present study, whole extract was used which may not be as genotoxic as the neutral fraction. In addition, the duration of treatment may be important for potential genotoxicity of airborne particles in in vivo situations. It may be observed that the amount and type of mutagens in airborne particles may vary with environmental and meteorological conditions. To properly evaluate the potential genotoxicity and carcinogenicity of air particulate extract in vivo, chronic studies may have to be conducted. Also, other tissues, such as respiratory, as well as other genetic end-points, might be considered for future studies. Since the airborne particle extract caused doserelated SCEs in primary mouse bone marrow and spleen cell cultures, these systems can be used in genotoxicity studies of ambient airborne particles and other environmental complex mixtures. Also, these assays are potentially useful for in vivo and in vitro comparative cytogenetic studies and hence for evaluating the relative value of in vitro assays using the same cell types. In vitro assays assess genotoxic potential while in vivo assays assess the ability of the test compound to express this potential in the organ being tested in an intact animal (Ashby, 1983). The bone marrow and spleen cell data on airborne particles presented here support
the viewpoint that in vitro results are not always predictive of activity in vivo. The positive mutagenic response of airborne particulate extract with the Ames assay (Table 4) is in agreement with our previous reports (Krishna et al., 1984, 1985b). At a concentration of 5.36 mg airborne particulate extract per plate, the average h i s + revertants with and without $9 activation were 563 and 345, respectively. Comparable doserelated mutagenicity was noticed both in the presence ( r = 0.99) and in the absence (r = 0.95) of metabolic activation. A lower frequency of h i s + revertants was observed in the absence (about 8-fold over controls) than in the presence of $9 activation (about ll-fold over controls). However, the highest concentration of airborne particulate extract tested (16.09 mg per plate) yielded lower h i s + revertants in the Ames TA98 strain both with and without $9 activation indicating toxicity at this concentration. It may be noted that all concentrations of airborne particles that were positive in the Ames assay were also positive in the in vitro SCE assay with mouse bone marrow and spleen cells. Recently, good correlations between dose-response of h i s + revertants in Ames test and frequency of
TABLE 4 G E N E MUTATIONS I N D U C E D BY SOLVENT EXTRACT OF A I R B O R N E PARTICLES IN Salmonella typhimurium U S I N G AMES ASSAY a Airborne particles TA98 his + revertants per plate b -1-S.D. (mg per plate) Negative control c Positive control d 0.60 1.79 5.36 16.09 a
b c d e f
With $9 activation Without $9 activation 49.75 + 4.86
39.25 + 2.22
1584.00+80.96
514.50+33.83
115.00+17.42 242.25 + 30.31 563.50 + 38.02 451.50+ 55.31
e e ~ e,f
105.50+17.82 222.75 + 20.71 345.50 + 24.77 233.25 + 20.55
e ~ e e,r
The concentration of airborne particles indicated was based on original particle weight. Average of 2 Expts. with duplicate plates in each experiment. 0.1 ml dimethylsulfoxide per plate. 0.25 /zg 2-aminoanthracene (with $9 activation) and 0.1 #g trinitrofluorenone (without $9 activation) per plate. Significant at p < 0.01, over controls. Toxic to the S. typhimurium.
163 S C E s in C h i n e s e h a m s t e r l u n g (V79) cells a n d human lymphocytes after treatment with airborne particle extract have been demonstrated (Lockard et al., 1981; A l i n k et al., 1983; K r i s h n a et al., 1984). H i g h e r his + r e v e r t a n t s in t h e A m e s a s s a y w i t h $9 a c t i v a t i o n suggests t h e p r e s e n c e of promutagens requiring metabolic activation.
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