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Effects of diesel exhaust particles on chromosome aberration, sister chromatid exchange and morphological transformation in cultured mammalian cells
61
CancerLetters, (1988) 61-66 Elsevier Scientific Publishers Ireland Ltd.
Effects of diesel exhaust particles on chromosome aberration, sister chromatid exchange and morphological transformation in cultured mammalian cells M.M. Hasegawa*,Y.
Nishi**, H.Tsuda**,N.
Inui*** andK.
Morimotoa
Bio/ogica/ Research Centet, Central Research institute, Japan Tobacco inc., 23 Nakogi, Hatano, “Department of Hygiene and Preventive Medicine, Osaka University, School of Medicine, Nakanosi~a,
Kanagawa, and Kita-ku, Osaka
Gwnf (Received 20 May 1988) (Revision received 5 July 1988) (Accepted 7 July 1988)
Summary
clear clastogenic and transforming capabilities but that HD is weaker in this regard.
The ability of diesel exhaust particles (diesel tar) from light-duty (LD) and heavy-duty (HO) engines to induce chromosome aberrations, sister chromatid exchanges (SCEs) and mortransformations is examined. phoZogica1 Chinese hamster V79 cells were treated with LD and HD diesel tar for 3 h in order to anaaberrations and SCEs. lyze chromosome BALB/c 3T3 cells were used for the morphological transformation test. LD tar induced significant numbers of chromosome aberrations, whereas HD did not. Both LD and HD samples increased the number of SCEs in a dosewith LD being more dependent fashion, potent. In the transformation test, LD tar also induced a significant number of Type 111foci, while HD was only weakly active. The transformed cells isolated from these Type Ill foci produced tumors when injected into nude mice. These results show that LD possesses Correspondence to: M.M. Hasegawa. Present address: ‘Toxicology Research Laboratories, Japan Tobacco Inc., 23 Nakogi, Hatano, Kanagawa. 257 Japan; * ‘Life Science Research Laboratory, Japan Tobacco Inc., 6-Z Umegaoka, Midori-ku, Yokohama, Kanagawa, Japan;
* ’ ‘Pharmaceutical Research Laboratories, Japan Tobacco Inc., 6-Z Umegaoka, Midori-ku, Yokohama, Kanagawa, Japan. 03043835/88/$03.50
0 1988 Elsevier Scientific Publishers
Published and Printed in Ireland
Keywords: diesel exhaust particles; chrosister chromatid mosome aberration; exchange; morphological transformation. Introduction It has been found that mixtures extracted from diesel exhaust particles (diesel tar) are mutagenic without metabolic activation in the Ames tests [3,10,34,24] and that the mutagenic activity of diesel tar varies depending on a number of factors including engine type, fuel, collection method and extracting solvent used [1,2,17,22]. In mammalian cell culture systems it has been shown that diesel tar induces gene mutations and SCEs [5,9,18]. It has also been shown that treatment of BALB/c 3T3 cells with diesel tar caused the induction of some transformed foci although dose-related responses were not observed [6]. However, in these studies, the engine type was not given sufficient consideration. Since diesel engines can be classified into two types, LD and HD, it is natural to expect that the chemical components of the tar samples from each engine type are different and
Ireland Ltd.
62
have differing abilities with regard to the induction of cytogenetic damage in mammalian cells. In this study, we have compared the abiiity of LD and HD tar to induce chromosome aberrations and SCEs using Chinese hamster V79 ceils and morphological transformations using mouse BALB/c 3T3 cetls. Materials and methods
Diesel exhaust particles (diesel tar) were coilected with teflon-coated filters from two types of engines, HD (111, 6 cyciinders) and LD (1.81, 4 cylinders) which were operated on engine dynamometers at a torque of 35.4 kg * m and 1200 rev./min and a torque of 4 kg - m and 1700 rev./min, respectively. The particle collected with the filters were samples extracted with dichloromethane, which was then evaporated, and the samples were stored frozen at - 8O*C. The samples were dissolved in dimethyisulfoxide (DMSO) at a concentration of 100 mg/ml for the bioassays. Cell culture V79 Chinese hamster cells and BALB/c 3T3 A31-l-1 cells were grown in Eagle’s minimum essential medium (MEM) supplemented with 10% fetal bovine serum (GIBCO, U.S.A.) at 37*C under 5% CO, in air with 100% humidity. Chromosome aberration test V79 ceils were plated at a density of 2 X lo5 ceils/25 cm2 flask. On the following day the ceils were treated with chemicals in the culture medium for 3 h. Then the ceils were washed twice with Hanks’ salt solution and cuitured in a normal medium for 21 h. For the last 2 h of the culture period, the cells were treated with a 0.05 pg/ml dose of colcemid. Chromosome preparations were made by the usual drying method and stained with Giemsa. For each treatment, counts were made on 100 well-spread metaphases, and aberrations were classified according to the criteria of Savage [20] with a slight modification.
Assay ojsister chromatid exchanges V79 cells were plated and treated with chemicals in the same way as in the chromosome aberration test. After they were washed, the cells were cultured in a medium containing 5-bromo-2’-deoxyuridine at a concentration of 2 pg/mi for 24 h; the flask was wrapped in aluminum foil and kept in the dark. A modified fluorescence plus Giemsa technique (161 was used for differential staining of sister chromatids. For each treatment, the SCEs in 50 wellspread metaphases were analyzed. Transformation test Twenty-four hours after BALB/c 3T3 cells were plated onto 60 mm dishes at a density of 1 x lo4 ceils/dish, they were treated with chemicals for 3 days. Then they were washed with Hanks’ salt solution, refed with fresh medium and cultured for another 4 weeks. The medium was changed twice a week. At the end of the incubation period, the dishes were fixed with methanol, stained with Giemsa and scored for morphologically transformed foci (Type III) that showed dense, muitilayered, random orientation at any part of the edge of the foci [7]. For the measurement of ceil survival, 200 cells were plated onto a 60 mm dish, and were treated with chemicals in the same way as for the transformation test. After incubation for 10 days, the dishes were fixed and stained, and the number of colonies was counted. Results and discussion The results regarding the induction of chromosome aberrations in V79 cells by diesel tar are shown in Table 1. In the solvent control, the frequency of aberrant cells was always less than 3%, while in cells treated with a 5 pg/mi dose of ~-methyl-~‘~nitro-~-ni~osoguanidine (MNNG) as a positive control the percentage of aberrant cells reached about 40%, and a high incidence of exchanges was observed. In cultures treated with LD tar the number of aberrant cells increased in a dose-dependent manner, but a significant increase occurred only at the highest dose tested. On the other
63 Table 1.
Chromosome
Treatment
aberrations
Dose (rg/mf)
Experiment I DMSO (0.3%) MNNG
5
induced
by diesel tar in cultured Chinese
hamster V79 cells.
% cells with aberrations
No. of aberrations/100
No. of cells observed
0
0
0
53
55
1
3
55
167
3 8 11 17b
2 11 19 22
01 81 51 10 2
0 0 0 0
0 4 2 7
3 24 27 41
3 5 6 3
02 11 01 02
0 0 0 0
0 2 1 0
5 9 8 5
2
00
0
0
2
6
47
113
100 150 200 250 300
100 100 100 100 No metaphases
00
100
2
100
3Bb
27
3 5 lib
3 3 13
00 01 70
0 0 0
0 1 5
3 5 25
2 6 8
3 6 8
00 10 20
0 0 0
0 2 4
3 9 14
LD
100 150 200 250
100 100 100 No metaphases
HD
100 150 200 250
100 100 100 metaphases
“cg, chromatid gap; cb, chromatid bP< 0.01 by x2-test.
ex
46b
HD
5
Total no.
ib
100 100 100 100 100 No metaphases
MNNG
ig
3
100 150 200 250 300
3
cb
100
LD
Experiment II DMSO (0.3%)
cg
cells”
break; ig, isochromatid
hand, treatment with HD tar led to no significant increase of chromosome aberrations at any of the doses tested. The types of chromosome aberrations induced by diesel tar were mainly chromatid gaps and breaks. Figure 1 shows that the cells treated with 50-200 pg/ ml doses of LD and HD tar induced a significant number of SCEs dose-dependently. The number of SCEs increased at most 1.6-fold upon treatment of cells with diesel tar. In contrast a 5-fold increase in SCEs was observed in
gap; ib, isochromatid
33
0
break; ex, exchange.
cells treated with mitomycin C (MMC, 10 ng/ ml) as a positive control (Fig. 1). These results show that diesel tar is weakly clastogenic in V79 cells in vitro, and that the clastogenic potency of LD tar is higher than that of HD. Results on the induction of morphological transformations in BALB/c 3T3 cells by diesel tar are summarized in Table 2. 3-Methylcholanthrene (MCA, 2 pg/ml) used as a positive control induced morphologically transformed Type III foci at a high frequency while treat-
64
6
s 5
L
oi ,
-.-
u)
-#I-
HD
~MClO,nWmlz3~*l.l~ 1
0 EMSO
50
loo
150
200
Diesel tar (Pg/ml)
Fig. 1. The frequency of SCEs (mean f S.E.M. from 50 metaphases) in V79 cells induced by LD and HD tar.
Table 2.
Morphological
Treatment
transformation
Dose (yg/mf
of BALB/c Relative survival
ment with DMSO, a solvent control, yielded no Type III foci at all. No significant increase in the number of transformed foci was observed for lo-50 pg/ml doses of HD tar, although a 50 pg/ml dose showed severe cytotoxicity. On the other hand, treatment with 30 and 50 pg/ml doses of LD tar induced a significant number of Type III foci. To examine the tumorigenicity of the transformed cells, a number of Type III foci were isolated from cultures treated with LD and HD tar and also MCA and were injected subcutaneously into nude mice (BALB/c nu/nu) at a dose of lo6 cells per animal. These transformed cells produced tumors in nude mice at the site of injection, but untreated and untransformed cells did not even when the dose was as high as lo7 cells/animal (data not shown). In this paper, we have shown the LD tar exhibits clear clastogenic and transforming
3T3 ceils by diesel tar. Total no. of foci
No. of foci/dish
(%,) Experiment
I
DMSO (0.2%) MCA HD
Experiment
Total no. of dishes with foci/total no. of dishes
100
0
2
5.1
45
10 20 30 50
95.7 49.7 17.2 0.8
0
O/16
2.5@
16/l&
1 1 1 6
0.05 0.05 0.05 0.30
l/19 l/20 l/20 3/20
0
0
o/14
!I
100
DMSO (0.2%) MCA LD
2
20.2
37
3.3&
13/14’
10 20 30 50
90.5 74.0 57.3 10.5
6 1 4 6
0.32 0.07 0.24b 0.4@
4/19 l/14 4/17 6.15d
"P< 0.01 by t-test. bO.O1 < P< 0.05 by t-test. cP< 0.01 byX2-test. d0.01 < P < 0.05 by x*-test.
65
activity but that HD tar is weaker in this regard. As diesel tar is a mixture of many substances, its mutagenicity is thought to result from the collective action of several major mutagens. It may therefore be difficult to explain the difference in the biological effects of LD and HD tar only through the qualitative and quantitative analysis of the chemical components of the tar samples. Results from the Salmonella test system indicate that nitrated pyrenes are the principal components of diesel tar that show mutagenic activity [12,15,21,25]. In tests with cultured mammalian cells, it has been shown that 1-nitropyrene and dinitropyrenes increase the number of single-strand breaks in DNA [19] and the number of SCEs [ 111, and that dinitropyrenes induce drug-resistant mutations [4,13,23]. The finding obtained through HPLC analysis that LD tar contains more lnitropyrene than HD tar[8] is consistent with our present results that LD tar is more potent than HD tar.
7
8
9
10
11
12
References Chan, T.L., Lee, P.S. and Siak, J.S. (1981) Diesel-partculate collection for biological testing. Comparison of electrostatic precipitation anf filtration. Environ. Sci. Technol., 15,89-93. Clark, C.R., Royer, R.E., Brooks, A.L., McClellan, R.O., Marshall, W.F., Naman, T.M. and Seizinger, D.E. (1981) Mutagenicity of diesel exhaust particle extracts: influence of car type. Fundam. Appl. Toxicol., 1, 260-265. Claxton. L.D. and Barnes, H.M. (1981) The mutagenicity of diesel-exhaust particle extracts collected under smogchamber conditions using the Salmonella typhimurium test system. Mutat. Res., 88, 255-272. Cole, J., Arlett, C.F., Lowe, J. and Bridges, B.A. (1982) The mutagenic potency of l,%dinitropyrene in cultured mouse lymphoma cells. Mutat. Res.. 93, 213-220. Coulomb, H.M.. Gu, Z.W., Loireau, M.P. and Chouroulinkov, I. (1984) Effect of serum concentration on the cytotoxicity and sister chromatid exchange induction by a chemical carcinogen and by a diesel particulate extract in V79 hamster cells. Cancer Len., 22, 77-82. Curren, R.D.. Kouri, R.E., Kim, C.M. and Schectman, L.M. (1980) Mutagenic and carcinogenic potency of extracts from diesel related environmental emissions: simultaneous morphological transformation and mutagenesis in BALB/c 3T3 cells. In: Health Effects of Diesel Engine Emissions, Proceedings of an International Symposium. Vol. 2, pp. 861-873.
13
14
15
16
17
18
19
20
Kakunaga, T. (1985) Critical review of the use of established cell lines for in-vitro cell transformation. In: Transformation Assay of Established Cell Lines: Mechanisms and Application, pp. 55-73. Editor: T. Kakunaga and H. Yamasaki. IARC Scientific Publications No. 67, Oxford University Press, New York. Kawai, A., Kawai, S., Sakairi, K., Sasaki, S. and Mori, T. (1987) Health effects of diesel emissions - Part 4 Change of mutagenicity and PAHs. In: Abstracts of Technical Papers presented at 1987 JDAE Spring Convention, pp. 207 -210. (in Japanese). Mitchell, A.D., Evans, E.L., Jotz, M.M.. Riccio, E.S., Mortelmans, K.E. and Simmon, V.F. (1980) Mutagenic and carcinogenic potency of extracts of diesel and related environmental emissions: in vitro mutagenesis and DNA damage. In: Health Effects of Diesel Engine Emissions, Proceedings of an International Symposium, Vol. 2, pp. 810-842. Nachtman, J.P., Xu, X.B., Rappaport, S.M., Talcon, R.E. and Wei, E.T. (1981) Mutagenic activity in diesel exhaust particulates. Bull. Environ. Contam. Toxicol., 27, 463-466. Nachtman, J.P. and Wolff, S. (1982) Activity of nitropolynuclear aromatic hydrocarbons in the sister chromatid exchange assay with and without metabolic activation. Environ. Mutagen., 4, l-5. Nakagawa, R., Kitamori, S., Horikawa, K., Nakashima, K. and Tokiwa, H. (1983) Identification of dinitropyrenes in diesel-exhaust particles; their probable presence as the major mutagens. Mutat. Res., 124, 201-211 Nakayasu, M., Sakamoto, H., Wakabayashi, K., Terada, M., Sugimura, T. and Rosenkranz, H.S. (1982) Potent mutagenic activity of nitropyrenes on Chinese hamster lung cells with diphtheria toxin resistance as a selective marker. Carcinogenesis, 3,917-922. Ohnishi, Y., Kachi, K., Sato, K., Tahara, I., Takeyoshi, H. and Tokiwa, H. (1980) Detection of mutagenic activity in automobile exhaust. Mutat. Res., 77, 229-240. Pederson, T.C. and Siak, J.S. (1981) The role of nitroaromatic compounds in the direct-acting mutagenicity or diesel particle extracts. J. Appl. Toxicol., 1, 54-60. Perry, P. and Wolff, S. (1974) New Giemsa method for the differential staining of sister chromatids. Nature, 251, 156-158. Rappaport, S.M., Wang, Y.Y. and Wei, E.T. (1980) Isolation and identification of a direct-acting mutagen in diesel-exhaust particulates. Environ. Sci. Technol., 14, 1505-1509. Rudd, C.J. (1979) Diesel particulate extracts in cultured mammalian cells. In: Health Effects of Diesel Engine Emissions, Proceedings of an International Symposium, Vol. 1, pp. 385-403. Saito, K., Mita, S., Kamataki, T. and Kato, R. (1984) DNA single-strand breaks by nitropyrenes and related compounds in Chinese hamster V79 cells. Cancer Len., 24,121-127. Savage, J.R.K. (1976) Classification and relationships of
66
21
22
23
induced chromosomal structural changes, J Med. Genet., 13,103122. Schuetzle, D., Riley, T.L., Prater, T.J., Harvey, T.M. and Hunt, D.F. (1982) Analysis of nitrated polycyclic aromatic hydrocarbons in diesel particulates. Anal. Chem., 54, 265 -271. Siak, J.S., Chan, T.L. and Lee, P.S. (1981) Diesel particulate extracts in bacterial test systems. Environ. Int., 5,243-248. Takayama, S., Tanaka, M., Katoh, Y., Terada, M. and
24
25
Sugimura, T. (1983) Mutagenicity of nitropyrenes in Chinese hamster V79 cells. Gann. 74, 338-341. Wei, E.T., Wang, Y.Y. and Rappaport, S.M. (1980) Diesel emissions and the Ames test: a commentary. J. Air. Pollut. Control Assoc., 30, 267-271. Xu, X.B., Nachtman, J.P., Jin, Z.L., Wei, E.T., Rappaport, S.M. and Burlingame, A.L. (1982) Isolation and identification of mutagenic nitro-PAH in diesel-exhaust particulates. Anal. Chim. Acta, 136, 163-174.
CancerLetters, (1988) 61-66 Elsevier Scientific Publishers Ireland Ltd.
Effects of diesel exhaust particles on chromosome aberration, sister chromatid exchange and morphological transformation in cultured mammalian cells M.M. Hasegawa*,Y.
Nishi**, H.Tsuda**,N.
Inui*** andK.
Morimotoa
Bio/ogica/ Research Centet, Central Research institute, Japan Tobacco inc., 23 Nakogi, Hatano, “Department of Hygiene and Preventive Medicine, Osaka University, School of Medicine, Nakanosi~a,
Kanagawa, and Kita-ku, Osaka
Gwnf (Received 20 May 1988) (Revision received 5 July 1988) (Accepted 7 July 1988)
Summary
clear clastogenic and transforming capabilities but that HD is weaker in this regard.
The ability of diesel exhaust particles (diesel tar) from light-duty (LD) and heavy-duty (HO) engines to induce chromosome aberrations, sister chromatid exchanges (SCEs) and mortransformations is examined. phoZogica1 Chinese hamster V79 cells were treated with LD and HD diesel tar for 3 h in order to anaaberrations and SCEs. lyze chromosome BALB/c 3T3 cells were used for the morphological transformation test. LD tar induced significant numbers of chromosome aberrations, whereas HD did not. Both LD and HD samples increased the number of SCEs in a dosewith LD being more dependent fashion, potent. In the transformation test, LD tar also induced a significant number of Type 111foci, while HD was only weakly active. The transformed cells isolated from these Type Ill foci produced tumors when injected into nude mice. These results show that LD possesses Correspondence to: M.M. Hasegawa. Present address: ‘Toxicology Research Laboratories, Japan Tobacco Inc., 23 Nakogi, Hatano, Kanagawa. 257 Japan; * ‘Life Science Research Laboratory, Japan Tobacco Inc., 6-Z Umegaoka, Midori-ku, Yokohama, Kanagawa, Japan;
* ’ ‘Pharmaceutical Research Laboratories, Japan Tobacco Inc., 6-Z Umegaoka, Midori-ku, Yokohama, Kanagawa, Japan. 03043835/88/$03.50
0 1988 Elsevier Scientific Publishers
Published and Printed in Ireland
Keywords: diesel exhaust particles; chrosister chromatid mosome aberration; exchange; morphological transformation. Introduction It has been found that mixtures extracted from diesel exhaust particles (diesel tar) are mutagenic without metabolic activation in the Ames tests [3,10,34,24] and that the mutagenic activity of diesel tar varies depending on a number of factors including engine type, fuel, collection method and extracting solvent used [1,2,17,22]. In mammalian cell culture systems it has been shown that diesel tar induces gene mutations and SCEs [5,9,18]. It has also been shown that treatment of BALB/c 3T3 cells with diesel tar caused the induction of some transformed foci although dose-related responses were not observed [6]. However, in these studies, the engine type was not given sufficient consideration. Since diesel engines can be classified into two types, LD and HD, it is natural to expect that the chemical components of the tar samples from each engine type are different and
Ireland Ltd.
62
have differing abilities with regard to the induction of cytogenetic damage in mammalian cells. In this study, we have compared the abiiity of LD and HD tar to induce chromosome aberrations and SCEs using Chinese hamster V79 ceils and morphological transformations using mouse BALB/c 3T3 cetls. Materials and methods
Diesel exhaust particles (diesel tar) were coilected with teflon-coated filters from two types of engines, HD (111, 6 cyciinders) and LD (1.81, 4 cylinders) which were operated on engine dynamometers at a torque of 35.4 kg * m and 1200 rev./min and a torque of 4 kg - m and 1700 rev./min, respectively. The particle collected with the filters were samples extracted with dichloromethane, which was then evaporated, and the samples were stored frozen at - 8O*C. The samples were dissolved in dimethyisulfoxide (DMSO) at a concentration of 100 mg/ml for the bioassays. Cell culture V79 Chinese hamster cells and BALB/c 3T3 A31-l-1 cells were grown in Eagle’s minimum essential medium (MEM) supplemented with 10% fetal bovine serum (GIBCO, U.S.A.) at 37*C under 5% CO, in air with 100% humidity. Chromosome aberration test V79 ceils were plated at a density of 2 X lo5 ceils/25 cm2 flask. On the following day the ceils were treated with chemicals in the culture medium for 3 h. Then the ceils were washed twice with Hanks’ salt solution and cuitured in a normal medium for 21 h. For the last 2 h of the culture period, the cells were treated with a 0.05 pg/ml dose of colcemid. Chromosome preparations were made by the usual drying method and stained with Giemsa. For each treatment, counts were made on 100 well-spread metaphases, and aberrations were classified according to the criteria of Savage [20] with a slight modification.
Assay ojsister chromatid exchanges V79 cells were plated and treated with chemicals in the same way as in the chromosome aberration test. After they were washed, the cells were cultured in a medium containing 5-bromo-2’-deoxyuridine at a concentration of 2 pg/mi for 24 h; the flask was wrapped in aluminum foil and kept in the dark. A modified fluorescence plus Giemsa technique (161 was used for differential staining of sister chromatids. For each treatment, the SCEs in 50 wellspread metaphases were analyzed. Transformation test Twenty-four hours after BALB/c 3T3 cells were plated onto 60 mm dishes at a density of 1 x lo4 ceils/dish, they were treated with chemicals for 3 days. Then they were washed with Hanks’ salt solution, refed with fresh medium and cultured for another 4 weeks. The medium was changed twice a week. At the end of the incubation period, the dishes were fixed with methanol, stained with Giemsa and scored for morphologically transformed foci (Type III) that showed dense, muitilayered, random orientation at any part of the edge of the foci [7]. For the measurement of ceil survival, 200 cells were plated onto a 60 mm dish, and were treated with chemicals in the same way as for the transformation test. After incubation for 10 days, the dishes were fixed and stained, and the number of colonies was counted. Results and discussion The results regarding the induction of chromosome aberrations in V79 cells by diesel tar are shown in Table 1. In the solvent control, the frequency of aberrant cells was always less than 3%, while in cells treated with a 5 pg/mi dose of ~-methyl-~‘~nitro-~-ni~osoguanidine (MNNG) as a positive control the percentage of aberrant cells reached about 40%, and a high incidence of exchanges was observed. In cultures treated with LD tar the number of aberrant cells increased in a dose-dependent manner, but a significant increase occurred only at the highest dose tested. On the other
63 Table 1.
Chromosome
Treatment
aberrations
Dose (rg/mf)
Experiment I DMSO (0.3%) MNNG
5
induced
by diesel tar in cultured Chinese
hamster V79 cells.
% cells with aberrations
No. of aberrations/100
No. of cells observed
0
0
0
53
55
1
3
55
167
3 8 11 17b
2 11 19 22
01 81 51 10 2
0 0 0 0
0 4 2 7
3 24 27 41
3 5 6 3
02 11 01 02
0 0 0 0
0 2 1 0
5 9 8 5
2
00
0
0
2
6
47
113
100 150 200 250 300
100 100 100 100 No metaphases
00
100
2
100
3Bb
27
3 5 lib
3 3 13
00 01 70
0 0 0
0 1 5
3 5 25
2 6 8
3 6 8
00 10 20
0 0 0
0 2 4
3 9 14
LD
100 150 200 250
100 100 100 No metaphases
HD
100 150 200 250
100 100 100 metaphases
“cg, chromatid gap; cb, chromatid bP< 0.01 by x2-test.
ex
46b
HD
5
Total no.
ib
100 100 100 100 100 No metaphases
MNNG
ig
3
100 150 200 250 300
3
cb
100
LD
Experiment II DMSO (0.3%)
cg
cells”
break; ig, isochromatid
hand, treatment with HD tar led to no significant increase of chromosome aberrations at any of the doses tested. The types of chromosome aberrations induced by diesel tar were mainly chromatid gaps and breaks. Figure 1 shows that the cells treated with 50-200 pg/ ml doses of LD and HD tar induced a significant number of SCEs dose-dependently. The number of SCEs increased at most 1.6-fold upon treatment of cells with diesel tar. In contrast a 5-fold increase in SCEs was observed in
gap; ib, isochromatid
33
0
break; ex, exchange.
cells treated with mitomycin C (MMC, 10 ng/ ml) as a positive control (Fig. 1). These results show that diesel tar is weakly clastogenic in V79 cells in vitro, and that the clastogenic potency of LD tar is higher than that of HD. Results on the induction of morphological transformations in BALB/c 3T3 cells by diesel tar are summarized in Table 2. 3-Methylcholanthrene (MCA, 2 pg/ml) used as a positive control induced morphologically transformed Type III foci at a high frequency while treat-
64
6
s 5
L
oi ,
-.-
u)
-#I-
HD
~MClO,nWmlz3~*l.l~ 1
0 EMSO
50
loo
150
200
Diesel tar (Pg/ml)
Fig. 1. The frequency of SCEs (mean f S.E.M. from 50 metaphases) in V79 cells induced by LD and HD tar.
Table 2.
Morphological
Treatment
transformation
Dose (yg/mf
of BALB/c Relative survival
ment with DMSO, a solvent control, yielded no Type III foci at all. No significant increase in the number of transformed foci was observed for lo-50 pg/ml doses of HD tar, although a 50 pg/ml dose showed severe cytotoxicity. On the other hand, treatment with 30 and 50 pg/ml doses of LD tar induced a significant number of Type III foci. To examine the tumorigenicity of the transformed cells, a number of Type III foci were isolated from cultures treated with LD and HD tar and also MCA and were injected subcutaneously into nude mice (BALB/c nu/nu) at a dose of lo6 cells per animal. These transformed cells produced tumors in nude mice at the site of injection, but untreated and untransformed cells did not even when the dose was as high as lo7 cells/animal (data not shown). In this paper, we have shown the LD tar exhibits clear clastogenic and transforming
3T3 ceils by diesel tar. Total no. of foci
No. of foci/dish
(%,) Experiment
I
DMSO (0.2%) MCA HD
Experiment
Total no. of dishes with foci/total no. of dishes
100
0
2
5.1
45
10 20 30 50
95.7 49.7 17.2 0.8
0
O/16
2.5@
16/l&
1 1 1 6
0.05 0.05 0.05 0.30
l/19 l/20 l/20 3/20
0
0
o/14
!I
100
DMSO (0.2%) MCA LD
2
20.2
37
3.3&
13/14’
10 20 30 50
90.5 74.0 57.3 10.5
6 1 4 6
0.32 0.07 0.24b 0.4@
4/19 l/14 4/17 6.15d
"P< 0.01 by t-test. bO.O1 < P< 0.05 by t-test. cP< 0.01 byX2-test. d0.01 < P < 0.05 by x*-test.
65
activity but that HD tar is weaker in this regard. As diesel tar is a mixture of many substances, its mutagenicity is thought to result from the collective action of several major mutagens. It may therefore be difficult to explain the difference in the biological effects of LD and HD tar only through the qualitative and quantitative analysis of the chemical components of the tar samples. Results from the Salmonella test system indicate that nitrated pyrenes are the principal components of diesel tar that show mutagenic activity [12,15,21,25]. In tests with cultured mammalian cells, it has been shown that 1-nitropyrene and dinitropyrenes increase the number of single-strand breaks in DNA [19] and the number of SCEs [ 111, and that dinitropyrenes induce drug-resistant mutations [4,13,23]. The finding obtained through HPLC analysis that LD tar contains more lnitropyrene than HD tar[8] is consistent with our present results that LD tar is more potent than HD tar.
7
8
9
10
11
12
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