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Genotoxicity of 1-nitropyrene and 2,4,7-trinitro-9-fluorenone to mammalian cells
Mutation Research, 240 (1990) 73-81
73
Elsevier MUTGEN 01506
Genotoxicity of 1-nitropyrene and 2,4,7-trinitro-9-fluorenone to mammalian cells H. Rodriguez a and G.L. Murison 2 Biology Department, Boston University, 2 Cummington St., Boston, MA 02215 (U.S.A.) and e Department of Biological Sciences, Florida International University, University Park, Miami, FL 33199 (U.S.A.)
(Received 20 March 1989) (Revision received19 June 1989) (Accepted 28 August 1989)
Keywords: Genotoxicity;Nitroarenes; Mammaliancells
Summary Nitroarenes are ubiquitous environmental pollutants displaying potent genotoxicity in bacterial and mammalian cells. In this study, 2,4,7-trinitro-9-fluorenone (TNF) was more potent than 1-nitropyrene (1-NP) in eliciting genotoxic responses in 4 mammalian cell lines. All 4 cell types were capable of activating the nitroarenes, since no special incubation conditions were required. Inhibition of normal DNA synthesis and cytotoxicity were significantly increased with TNF in a dose range of 0.2-5 /~g/ml for human teratocarcinoma (PAl) cells, mouse Sertoli (TM4) cells, rat hepatoma (RL12) cells, and human-Chinese hamster ovary (CHO-K1) cells. For 1-NP, a dose range of 1 0 - 2 0 / t g / m l was required to achieve comparable results for the respective cell lines. Only the RL12 and CHO-K1 cells showed recovery of normal DNA synthesis when TNF or 1-NP was removed from the medium. The other cell types showed little or no recovery up to 42 h after removal of the nitroarene. In exclusively studying TNF, the induction of sister-chromatid exchanges (SCEs) and a delay in cell cycle as monitored by harlequin chromosomes, were observed at a concentration range of 0.003-0.2 /xg/ml in PAl, TM4, and RL12 cells. In CHO-K1 cells, TNF at 0.001-1/~g/ml was clearly mutagenic at the hprt locus.
Nitroarenes are common environmental contaminants that exhibit genotoxic activity (Rosenkranz and Mermelstein, 1983, 1985; Tokiwa and Ohnishi, 1986). These compounds, which have been found in photocopier toners (Rosenkranz et al., 1980), diesel exhaust (Salmeen et al., 1982), urban air particulates (Tokiwa et al., 1983) and coal fly ash (Wei et al., 1982), arise from atmo-
Correspondence: Dr. H. Rodriguez,BiologyDepartment, Boston University,2 CummingtonSt., Boston,MA 02215 (U.S.A.).
spheric reactions of polycyclic aromatic hydrocarbons (PAHs) with HNO3 and NO: (Hirayama et al., 1983) and from incomplete combustion processes (Yergey et al., 1982). Nitroarenes are potent direct-acting bacterial mutagens (Rosenkranz and Mermelstein, 1983). In bacterial assays, the mutagenicity is dependent upon enzymatic reduction of the nitro moiety (McCoy et al., 1981; Heflich et al., 1985). In Salmonella t y p h i m u r i u m , these compounds are activated to intermediates capable of forming adducts with the cellular DNA (Rosenkranz, 1982).
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74 In contrast to the substantial body of literature concerning the mutagenicity of nitroarenes in Salmonella strains (Rosenkranz and Mermelstein, 1983), the reports based on mammalian (rodent and human) cell assays are less clear and consistent. For instance, Howard and Beland (1982) and Beland et al. (1986) reported that transformation by 1-NP of normal human diploid fibroblasts to a state of anchorage-independent cells, as well as cellular invasiveness, occurred only under an argon atmosphere with the inclusion of xanthine oxidase. By contrast, Patton et al. (1986) induced mutagenic and cytotoxic effects in diploid human fibroblasts with 1-NP in the presence of oxygen and absence of xanthine oxidase. In this paper, we have elected to study the potency of 2 nitroarenes, T N F and 1-NP (in the absence of $9 and presence of oxygen), on 4 different mammalian cell fines using 6 different assay systems. It has been shown that trinitro (as opposed to mononitro and dinitro) compounds generally induce the highest levels of mutagenic and genotoxic actions in various bacteria (Rosenkranz and Mermelstein, 1983) and cultured mammalian cells (Li and Dutcher, 1983). Thus, we have chosen to study the effects of T N F on 4 mammalian cells and compare the results to those produced by 1-NP, a weak mammalian nitroarene (Li and Dutcher, 1983). Part of the conflicting data may be due to different incubation conditions, and the use of different tissue or species. In order to examine whether the discrepancies are due to tissue or species differences, the following cell lines were used: a human teratocarcinoma cell (PAl), a mouse Sertoli cell (TM4), a rat hepatocyte cell (RL12) and a human-Chinese hamster ovarian cell (CHO-K1). Each cell type is useful for a unique purpose: PAl cells are a stable human diploid cell line and have been used previously for mutation assays (Huberman et al., 1984), TM4 cells have characteristics that are consistent with those seen in primary cultures of Sertoli cells (Mather, 1980), RL12 cells are known to metabolize a number of PAHs and might be capable of metabolizing nitroarenes (Murison et al., 1984), and CHO-K1 cells are a stable diploid cell line that has been used previously in mutation assays (Waldren et al., 1979). In doing such a compara-
tive study, we hope to resolve some of the conflicting data on the effects of nitroarenes on various rodent and human cells. Materials and methods
Chemicals 2,4,7-Trinitro-9-fluorenone ( T N F ; purity > 98% as detected by HPLC) and 1-nitropyrene (1-NP; purity > 98% as detected by HPLC) were obtained from the Fluka Chemical Company, Ronkonkoma, NY, while 5-bromodeoxyuridine (BrdUrd), colchicine, and 6-thioguanine (TG) were obtained from the Sigma Chemical Company, St. Louis, MO. The nitroarenes and T G were dissolved in dimethyl sulfoxide (DMSO) prior to their dilution into the culture medium. The BrdUrd and colchicine were dissolved in Dulbecco's phosphate buffer solution (PBS). Cells The characteristics of the PAl clonal isolate obtained from a human teratocarcinoma were described previously (Huberman et al., 1984). The mouse Sertofi cell line TM4 was obtained from primary cultures of Sertoli cell-enriched preparations from normal testis of l l - 1 3 - d a y - o l d B a l b / c n u / + mice (Mather, 1980). The rat hepatoma cell line RL12 had been shown previously to metabolize a number of polycyclic aromatic hydrocarbons (Murison et al., 1984). The human-Chinese hamster cell line CHO-K1 was obtained from fusion of human amniotic fluid fibroblasts and the gly-A mutant of the Chinese hamster ovary cell. This cell contains the standard chromosomes of the CHO-K1 cell plus human chromosome 11 (Puck et al., 1971). All cell lines were cultured routinely in a humidified atmosphere (5% CO 2 in 95% air) in D M E M medium supplemented with 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100/~g/ml) (Gibco, Grand Island, NY). In the cytotoxicity and mutagenicity assays where cloning densities were determined, Ham's F12 (Gibco) supplemented with 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 /~g/ml) was used in place of DMEM. Inhibition of normal DNA synthesis assay All cell lines were subcultured with 0.05% trypsin (Gibco) and transferred to 35-ram dishes
75 (3 dishes/dose) at 1 x 106 cells/dish. After 12-24 h, the cells were treated with the desired concentration of T N F or 1-NP dissolved in DMSO (Mallinckrodt) to a final concentration of DMSO not exceeding 0.05%. After 45 min, [Me3H]thymidine ( 1 / t C i / m l ; 60 Ci/mmole; ICN Radiochemicals, Irvine, CA) was added to each dish. After 3 h of exposure, cells were harvested and lysed overnight in 1 N N H 4 O H and 0.2% Triton X-100 (Fisher Scientific, Pittsburgh, PA). A small aliquot (0.1 ml) of the lysate was removed for total D N A content determination by the use of a T K O 100 DNA fluorometer (Hoefer Scientific Instruments). The rest of the lysate was precipitated with cold 10% trichloroacetic acid, 3% hydrochloric acid and filtered onto G F / A glass microfiber filters (Whatman). The filters were oven dried and placed into a liquid scintillation vial that contained 5 ml of Merrit liquid scintillation cocktail (Isolab). The counts per minute were registered on an LS 100 counter (Beckman).
DNA synthesis recovery assay All cell lines were subcultured with 0.05% trypsin and transferred to 35-mm dishes (3 dishes/dose) at 1 x 10 6 cells/dish. After 12-24 h, the cells were treated with the desired concentration of T N F or 1-NP dissolved in DMSO to a final concentration of DMSO not exceeding 0.05%. After 45 rain, the medium was removed, and fresh culture medium was added. After a 21- and 42-h recovery period, [ Me- 3H]thymidine (1/~ Ci/ml; 60 Ci/mmole) was added. After 3 h of exposure, cells were harvested and lysed overnight in 1 N N H 4 O H and 0.2% Triton X-100. A small aliquot of the lysate was removed for total DNA content determination by the use of a T K O 100 DNA fluorometer. The rest of the lysate was precipitated with cold 10% trichloroacetic acid and 3% hydrochloric acid and filtered onto G F / A glass microfiber filters. The filters were oven dried and placed into a liquid scintillation vial that contained 5 ml of Merrit liquid scintillation cocktail. The counts per minute were registered on an LS 100 counter. Cytotoxicity assay The PAl, TM4, and CHO-K1 cells were subcultured with 0.05% trypsin and transferred to
60-mm dishes (3 dishes/dose) at 500 or 600 cells/dish. After 12-24 h, the cells were treated with the desired concentration of T N F or 1-NP dissolved in DMSO. After 45 min, the medium was removed, and fresh culture medium was added. After 7 days of incubation, colonies were counted by fixing the cells with 70% ethanol and staining with 0.1% crystal violet (Sigma).
Sister-chromatid exchange/harlequin chromosome assay The PAl, RL12, and TM4 cells were subcultured with 0.05% trypsin and transferred to 35-mm dishes at 5 X 10 s cells/dish. After 12-24 h, the cells were treated with the desired concentration of T N F dissolved in DMSO. The culture medium added contained 0.2 m M BrdUrd. The cells were grown in the dark for at least 2 cell cycles (approximately 36 h). Colchicine was added at 0.01 m g / m l for the last 2 h of incubation. The cells were harvested, suspended in hypotonic KC1 (0.075 M), and kept at room temperature for 15 rain. The cells were then fixed in at least 3 changes of glacial acetic acid:methanol solution (1:3), and the cell suspension was dropped onto clean glass slides. 2-day-old slides, kept on a 5 5 ° C slide warmer, were then processed for SCE and percent harlequin analysis by a modification of the hot salt Giemsa technique (Korenberg and Freedlender, 1974). A minimum of 15 sets of chromosomes were scored for SCE/harlequin analysis. Mutagenicity assay CHO-K1 cells were subcultured with 0.05% trypsin and transferred to 100-mm dishes at 2 x 106cells/dish. After 12-24 h, the cells were treated with the desired concentration of T N F dissolved in DMSO. After 45 rain, the medium was replaced with fresh culture medium. After a 7-10-day expression period, cells were subcultured onto 60-mm dishes (10 dishes/dose) at 4 X 10 4 cells/dish. The culture medium contained 10 /~M TG. Additionally, cells were subcultured onto 60-mm dishes (3 dishes/dose) at 500 cells/dish for plating efficiency and cytotoxicity determination. After 7 days, the colonies were counted by fixing the cells with 70% ethanol and staining with 0.1% crystal violet.
76 Results
Inhibition of normal DNA synthesis Fig. 1A shows that inhibition of normal D N A synthesis was greatly increased in all cell types. A 2-way analysis of variance indicated that significant differences in inhibition of normal D N A synthesis between the 4 cell types was evident ( p < 0.0001). In all 4 cell types, the final concentration of T N F required to give 50% inhibition of normal DNA synthesis was 5 /~g/ml. Normal D N A synthesis was inhibited greater than 99% in all 4 cell types when T N F was added at a final concentration of 30 # g / m l . Fig. 1B shows that significant differences in inhibition of normal D N A synthesis were observed between the 4 cell types ( p < 0.0001) when exposed to 1-NP. In all 4 cell types, the final concentration of 1-NP required to give 50% inhibition of normal D N A synthesis was 20/~g/ml. At 30 /zg/ml, 1-NP inhibited normal D N A synthesis no greater than 91% in all 4 cell lines. Therefore, a significant difference was observed between 1-NP and T N F in the degree of inhibition of normal D N A synthesis. This indicates that 1-NP was a less potent inhibitor of normal D N A synthesis than T N F under the present conditions.
DNA synthesis recovery Exposure time of the cells to T N F was based on our previous studies, which showed that 45 rain of exposure produced the same amount of inhibition of normal D N A synthesis as did 3 h and 45 min of exposure (see Figs. 1 and 2). Fig. 2A shows that significant differences in inhibition of normal DNA synthesis were observed between the cell types ( p < 0.0001) when allowed to recover from T N F 21 h after being exposed to the nitroarene. When T N F was at a final concentration of 5 /~g/ml (dosage required for 50% inhibition), both the RL12 and CHO-K1 cells showed recovery from the initial inhibition, while the PAl and TM4 cells did not. Also at this dosage, the RL12 cells showed a stimulation in normal D N A synthesis. Similar results were obtained when the 4 cell types were allowed 42 h of recovery (data not shown). Fig. 2B shows the recovery of normal D N A synthesis after exposure to 1-NP. A significant
difference in inhibition of normal D N A synthesis was observed between the 4 cell types ( p < 0.0001). TM4 cells showed a stimulation in normal DNA synthesis when exposed to 1-NP at a final concentration of 0.2/~g/rnl, while the RL12 cells also exhibited such an effect, but at 5 /~g/ml. When 1-NP was at a final concentration of 20 /~g/ml (dosage required for 50% inhibition), all 4 cell lines exhibited recovery from the initial inhibition. When the cells were allowed 42 h of recovery (data not shown), no differences were observed from those seen at 21 h.
Cytotoxicity Fig. 3 shows that a significant difference ( p < 0.0001) in cytotoxic response to T N F was observed between the PAl, TM4, and CHO-K1 cell types. Overall, the CHO-K1 cells were less affected by the cytotoxic effect of T N F than the PAl and TM4 cells. The cytotoxic potency of 1-NP was less than that for T N F when tested on the PAl, TM4 and CHO-K1 cells (Fig. 4). A significant difference ( p < 0.0001) in cytotoxic response to 1-NP was observed between the 3 cell types. Overall, the TM4 cells were less affected by 1-NP, than the PAl and CHO-K1 ceils. To achieve at least a 50% cytotoxic effect for the PAl, TM4, and CHO-K1 ceils, 1-NP must be added at a final concentration of 25 /~g/ml as opposed to 1 /~g/ml as seen for T N F (Fig. 3). This indicates that T N F was 25 times more cytotoxic than 1-NP. At this point, observing that T N F was far more potent than 1-NP, we decided to focus our studies solely on TNF. Thus 1-NP was not tested for SCEs nor mutagenicity.
Sister-chromatid exchange~harlequin chromosomes Table 1 shows that T N F induced SCEs in PAl, TM4, and RL12 cells. A significant correlation was observed between the dosage of T N F and the number of SCEs per chromosome for the 3 cell types ( p < 0.0001). Significant variations in SCEs per chromosome were detected between the 3 cell types ( p < 0.0001). For the PAl, TM4, and RL12 cells, when the final concentration of T N F exceeded 0.2/~g/ml, cell killing (lysis) was observed. The production of harlequin chromosomes may be used to determine the length of a cell cycle. The
77 effects that T N F had on the cell cycle of the PAl, TM4, and RL12 cells are shown in Table 1. Although the cell cycle of all 3 cell types slowed down with increasing dosage of TNF, the cell cycle of the PAl and TM4 cells slowed down to a greater extent than that seen for the RL12 cells. A correlation between cell cycle and dosage was detected ( p < 0.0001) for the 3 cell types. Significant variations in inhibition of cell cycle were observed between the 3 cell types ( p < 0.0001).
100
10
Mutagenicity
tn cD .c
E < Z
0.1
f
20C
i
|
i
i
i
tx
i
I-NP
Fig. 5 shows the dose-response relationship between mutagen concentrations and mutation frequencies at the hprt locus. Although the frequency of spontaneous mutants for the C H O K1 cells was 12 m u t a n t s / 1 0 5 survivors, the mutant frequency due to T N F at a dosage of 0.001/~g/ml, was 49 mutants/105 survivors. When T N F was at a final concentration of 0.1 # g / m l , the mutant frequency was 80 mutants/105 survivors, and at 1 /~g/ml, T N F induced a mutant frequency of 168 mutants/105 survivors. These data indicate that T N F produced a 4-14-fold increase in mutant frequency over the control.
r
Discussion &
I(X:
0.2
I 5
I 10
I 15 Dose
I 20
I 25
I 30
(,ug / m l )
Fig. 1. (A) DNA synthesis in PAl ([2), TM4 (zx),RL12 (©) and CHO-K1 (I) calls exposed to TNF. The data are presented as the means of triplicates compared to controls without TNF. (B) DNA synthesis in PAl (t3), TM4 (zx), RL12 (o) and CHO-K1 (I) cells exposed to 1-NP. The data are presented as the means of triplicates compared to controls without 1-NP.
In this study, we have shown that T N F was more potent than 1-NP as an inhibitor of normal D N A synthesis. All cell types showed maximal inhibition of normal D N A synthesis in the presence of T N F , while with 1-NP, all 4 cell types showed much less inhibition. Since the experiments were performed in the absence of $9, all 4 cell types must be capable of activating the nitroarenes. In a similar study by Li and Dutcher (1983) using C H O cells, they also reported that trinitro compounds such as 1,3,6-TNP are more potent than 1-NP, as seen by higher levels of mutagenic and genotoxic effects. Both the T M 4 and RL12 cells showed an increase in D N A synthesis over the control rate when exposed to 1-NP at a low concentration of 0.2 /xg/ml. A plausible explanation for such a response might be that this stimulus may be due to D N A repair. Since the activation of nitroarenes by m a m m a l i a n cells forms intermediates capable of forming adducts with the cellular D N A (Patton
78
100 ~
et al., 1986), m a y b e in this study, the cells u n d e r went some form of D N A repair at the low dosages of 1-NP. This would thereby a c c o u n t for the in-
Cytotoxicity
T.
200
TNFA
O 0.01
0.1 Doseof TNF ( }Jg/rnl)
1
Fig. 3. Sur4val of P A l (rn), T M 4 (z~) and C H O - K I ( I ) cells
exposed to TNF. The data are presented as the means of triplicates compared to controls without TNF. Cloning efficiencies were as follows: PAl, 9%; TM4, 21% and CHO-K1, 91%.
10 '
~ ~ 0
[]
in
2OO
1-NP
Q
B
o
°
crease in D N A synthesis over the controls without 1-NP at the low doses. Also, even though it was with a different cell type, 1-NP has b e e n reported to simulate repair synthesis in freshly explanted h u m a n b r o n c h u s epithelial cells (Kawachi, 1982). It is o b v i o u s that studies o n D N A repair m u s t be p e r f o r m e d o n these 4 cell types so as to resolve whether a repair m e c h a n i s m is present or not. W h e n the cells were allowed u p to 42 h to recover from exposure to T N F , only the RL12 a n d C H O - K 1 cells showed some recovery, while the P A l a n d T M 4 cells showed n o recovery within this time period. W h e n the cells were allowed up to 42 h to recover from exposure to 1-NP, the RL12 a n d C H O - K 1 cells showed a greater recovery t h a n the P A l a n d T M 4 cells. This was evident w h e n 1-NP was a d m i n i s t e r e d at 20 a n d 30 / s g / m l . Also, the T M 4 a n d RL12 cells showed this increase i n p e r c e n t D N A synthesis over the con-
10
1
0.2
'
5
o
1
i
i
15 20 Dose (#g/rnl)
21
5
i
30
Fig. 2. (A) DNA synthesis in PAl (C3),TM4 (zx), RL12 (o) and CHO-K1 (11) cells exposed to TNF for 45 min and then allowed 21 h to recover. The data are presented as the means of triplicates compared to controls without TNF. (B) DNA synthesis in PAl (rq), TM4 (zx), RL12 (o) and CHO-K1 (11) cells exposed to 1-NP for 45 rain and then allowed 21 h to recover. The data are presented as the means of triplicates compared to controls without 1-NP.
79
2°°I
Cytotoxicity
TABLE 1 SCEs P E R C H R O M O S O M E CELLS EXPOSED T O T N F
IN PAl, T M 4 A N D RL12
The data are presented as the m e a n ± S.D.; n = 20, except for RL12 where n =15. Also, harlequin chromosomes in PAl, T M 4 and RL12 cells exposed to TNF. Dose of TNF
100
SCEs/chromosome PAl
TM4
% harlequin RL12
PAl
T M 4 RL12
(t,g/w])
-5 .> >
b
(/3 10
1/
0.2
I
5
I
10
'
'
2i
15 20 5 Dose o f I - NP ( ~g / ml )
30
control
0.26+0.1 0.15+0.1 0.205-0.1 100
100
100
0.001 0.003
0.365-0.1 0.34+0.1 0.54-1-0.1 0.42+0.1 0.39+0.1
88.2 77.6
98.6 72.8
91.3
0.01 0.03
0.71+0.1 0.58+0.1 0.495-0.1 1.09+0.1 0.99+0.1 0.78+0.1
70.6 64.7
64.3 57.1
84.8 73.9
0.1 0.3
1.23+0.3 1.22+0.2 1.055-0.3 1.33+0.1 1.45+0.2 1.28+0.2
57.6 50.6
50.0 42.8
63.0 57.6
oxidase. When the TM4 cells were exposed to 1-NP at 0.2-15 # g / m l , cell growth was stimulated above control levels. To explain this stimulation in cell division, future studies on D N A synthesis/cell division need to be performed. Exposure of the cells to T N F proved to be much more cytotoxic than 1-NP. T N F was shown to be 25 times more cytotoxic than 1-NP. CHO-K1 cells were more
Fig. 4. Survival of P A l (D), T M 4 (zx) and CHO-K1 (I) cells exposed to 1-NP. The data are presented as the means of triplicates compared to controls without 1-NP. Cloning efficiencies were as follows: PAl, 17%; TM4, 13% and CHO-K1, 77%.
180 160
140
trol rate when exposed to 1-NP at a concentration range from 0.2 to 20 # g / m l . It has been shown that 1-NP is cytotoxic to diploid human fibroblasts (Patton et al., 1986; Beland et al., 1986). In this study, 1-NP was also cytotoxic to the PAl, TM4, and CHO-K1 cells. These results were obtained in the presence of oxygen and absence of xanthine oxidase. This was contrary to what has been reported previously by Howard and Beland (1982) and Beland et al. (1986) who reported that an oxygen-free atmosphere with the inclusion of xanthine oxidase was required to activate nitroarenes. By contrast, our results are similar to the results obtained by Patton et al. (1986) who were able to induce cytotoxic effects in diploid human fibroblasts with 1-NP in the presence of oxygen and absence of xanthine
LnO 120
c 10C L
80
~. so E 4C 20
/ &
o.ool
I
0.01 01.1 Dose of TNF ( }Jg/rnl )
110
Fig. 5. D o s e - r e s p o n s e relationship between mutagen concentrations and m u t a n t frequencies at the hprt locus in CHOK1 ( I ) cells. The cloning efficiency of the CHO-K1 cells at the time of selection (7-10-day expression period) was 65 %.
80 resistant to the c y t o t o x i c effect of T N F t h a n were the P A l a n d T M 4 cells. Both the RL12 a n d P A l cells have b e e n shown previously to give rise to SCEs when e x p o s e d to P A H s ( M u r i s o n et al., 1984). I n this study, the P A l , TM4, a n d RL12 cells were e x p o s e d exclusively to T N F a n d then processed for S C E a n a l y sis. A l t h o u g h all 3 cell types showed an increase in SCEs when exposed to T N F , differences in SCEs p e r c h r o m o s o m e were detected b e t w e e n the 3 cell types. Ishidate a n d O d a s h i m a (1977) have shown that carcinogens have the p o t e n t i a l to m a r k e d l y i n h i b i t cell growth. A l t h o u g h the cell cycle of all 3 cell types ( P A l , TM4, a n d RL12) were greatly inhibited b y e x p o s u r e to T N F , the cell cycle of the RL12 cells were slowed to a lesser extent t h a n that observed for the P A l a n d T M 4 cells. In this study, we have shown that T N F was a p o t e n t mutagen, a n d this was i n d i c a t e d b y the m u t a n t frequency p e r g g of mutagen. T h e results o b t a i n e d here were o b t a i n e d in the presence of oxygen a n d absence of x a n t h i n e oxidase. This was similar to the results o b t a i n e d b y P a t t o n et al. (1986) who i n d u c e d m u t a g e n i c effects in d i p l o i d h u m a n fibroblasts with 1-NP in the presence of oxygen a n d absence of x a n t h i n e oxidase. Thus, n o special i n c u b a t i o n c o n d i t i o n s were r e q u i r e d as app o s e d to that r e p o r t e d b y H o w a r d a n d B e l a n d (1982) a n d B e l a n d et al. (1986). I n conclusion, we have d e m o n s t r a t e d that T N F was significantly m o r e genotoxic t h a n 1 - N P in all 4 cell lines a n d significantly m u t a g e n i c to C H O - K 1 cells. Also, no special i n c u b a t i o n c o n d i t i o n s (oxygen-free a t m o s p h e r e with the inclusion of xanthine oxidase) were r e q u i r e d to activate the nitroarenes. A l l 4 cell lines, r e p r e s e n t a t i v e of 4 species a n d 3 different tissues, were c a p a b l e of activating b o t h nitroarenes, a n d showed similar qualitative responses b u t different q u a n t i t a t i v e responses to these 2 nitroarenes. F u r t h e r d e t e r m i n a tion of the n i t r o r e d u c t a s e levels in each cell line w o u l d p r o v i d e useful i n f o r m a t i o n for u n d e r s t a n d ing the basis of these results.
Acknowledgement This w o r k was s u p p o r t e d b y a g r a n t f r o m the N a t i o n a l I n s t i t u t e of E n v i r o n m e n t a l H e a l t h to Dr. G e r a l d L. Murison.
References Beland, F.A., M. Ribovich, P.C. Howard, R.H. Heflich, P. Kurian and G.E. Milo (1986) Cytotoxicity, cellular transformation and DNA adducts in normal human diploid fibroblasts exposed to 1-nitrosopyrene, a reduced derivative of the environmental contaminant, 1-nitropyrene, Carcinogenesis, 7, 1279-1283. Heflich, R.H., P.C. Howard and F.A. Beland (1985) 1-Nitrosopyrene: an intermediate in the metabolic activation of 1-nitropyrene to a mutagen in Salmonella typhimurium TA1538, Mutation Res., 149, 25-32. Hirayama, T., M. Nohara, T. Ando, M. Tanaka, K. Nagano and S. Fukui (1983) Mutagenicity of coal-pyrolyzed products and their photochemical reaction products with nitrogen oxides in Salmonella typhimurium TA98 and TA100, Mutation Res., 122, 273-278. Howard, P.C., and F.A. Beland (1982) Xanthine oxidase catalyzed binding of 1-nitropyrene to DNA, Biochem. Biophys. Res. Cornrnun., 104, 727-732. Huberman, E., C.K. McKeown, C.A. Jones and S. Murao (1984) Induction of mutations by chemical agents at the hypoxanthine guanine phosphoribosyltransferase locus in human epithelial teratoma cells, Mutation Res., 130, 127-137. lshidate, M., and S. Odashima (1977) Chromosome tests with 134 compounds on Chinese hamster cells in vitro - - A screening for chemical carcinogens, Mutation Res., 48, 337-354. Kawachi, T. (1982) Mutagenicity and carcinogenicity of nitropyrenes, in: D.E. Rickert (Ed.), The Toxicity of Nitroaromatic Compounds, Hemisphere Press, New York. Korenberg, J.R., and E.F. Freedlender (1974) Giemsa technique for the detection of sister chromatid exchanges, Chromosoma, 48, 355-360. Li, A.P., and J.S. Dutcher (1983) Mutagenicity of mono-, di-, and tri-nitropyrenes in Chinese hamster ovary cells, Mutation Res., 119, 387-392. Mather, J.P. (1980) Establishment and characterization of 2 distinct mouse testicular epithelial-cell lines, Biol. Reprod., 23, 243-252. McCoy, E.C., H.S. Rosenkranz and R. Mermelstein (1981) Evidence of the existence of a family of bacterial nitroreductases capable of activating nitrated polycyclics to mutagens, Environ. Mutagen, 3, 421-427. Murison, G.L., S.D. Hall and A.M. Lee (1984) Metabolic activation of procarcinogens to genotoxic products in cultured rat liver cells, Exp. Cell. Biol., 52, 355-360. Patton, J.D., V.M. Maher and J.J. McCormick (1986) Cytotoxic and mutagenic effects of 1-nitropyrene and 1-nitrosopyrene in diploid human fibroblasts, Carcinogenesis, 7, 89-93. Puck, T.T., P. Wuthier, C. Jones and F.T. Koa (1971) Genetics of somatic mammalian cells: lethal antigens as genetic markers for study of human linkage groups, Proc. Natl. Acad. Sci. (U.S.A.), 68, 3102-3106. Rosenkranz, H.S. (1982) Direct-acting mutagens in diesel exhausts: magnitude of the problem, Mutation Res., 101, 1-10.
81 Rosenkranz, H.S., and R. Mermelstein (1983) Mutagenicity and genotoxicity of nitroarenes: all nitro-containing chemicals were not created equal, Mutation Res., 114, 217-267. Rosenkranz, H.S., and R. Mermelstein (1985) The genotoxicity, metabolism and carcinogenicity of nitrated polycychc aromatic hydrocarbons, J. Environ. Sci. Health, 2, 221-272. Rosenkranz, H.S., E.C. McCoy, D.R. Sanders, M. Butler, D.K. Kiriazides and R. Mermelstein (1980) Nitropyrenes: isolation, identification, and reduction of mutagenic impurities in carbon black and toners, Science, 209, 1039-1043. Salmeen, I., A.M. Durisin, T.J. Prater, T. Riley and D. Schuetzle (1982) Contribution of 1-nitropyrene to direct-acting Ames assay mutagenicities of diesel particulate extracts, Mutation Res., 104, 17-23. Tokiwa, H., and Y. Ohnishi (1986) Mutagenicity and carcino-
genicity of nitroarenes and their sources in the environment, CRC C. R. Toxicol., 17, 23-60. Tokiwa, H., S. Kitamori, R. Nakagawa, K. Horikawa and L. Matamala (1983) Demonstration of a powerful mutagenic dinitropyrene in airborne particulate matter, Mutation Res., 121, 107-116. Waldren, C., C. Jones and T.T. Puck (1979) Measurement of mutagenesis in mammalian cells, Proc. Natl. Acad. Sci. (U.S.A.), 76, 1358-1362. Wei, C., O.G. Raabe and L.S. Rosenblatt (1982) Microbial detection of mutagenic nitro-organic compounds in filtrates of coal fly ash, Environ. Mutagen., 4, 249-258. Yergey, J.A., T.N. Risby and S.S. Lestz (1982) Chemical characterization of organic adsorbates on diesel particulate matter, Anal. Chem., 54, 354-357.
73
Elsevier MUTGEN 01506
Genotoxicity of 1-nitropyrene and 2,4,7-trinitro-9-fluorenone to mammalian cells H. Rodriguez a and G.L. Murison 2 Biology Department, Boston University, 2 Cummington St., Boston, MA 02215 (U.S.A.) and e Department of Biological Sciences, Florida International University, University Park, Miami, FL 33199 (U.S.A.)
(Received 20 March 1989) (Revision received19 June 1989) (Accepted 28 August 1989)
Keywords: Genotoxicity;Nitroarenes; Mammaliancells
Summary Nitroarenes are ubiquitous environmental pollutants displaying potent genotoxicity in bacterial and mammalian cells. In this study, 2,4,7-trinitro-9-fluorenone (TNF) was more potent than 1-nitropyrene (1-NP) in eliciting genotoxic responses in 4 mammalian cell lines. All 4 cell types were capable of activating the nitroarenes, since no special incubation conditions were required. Inhibition of normal DNA synthesis and cytotoxicity were significantly increased with TNF in a dose range of 0.2-5 /~g/ml for human teratocarcinoma (PAl) cells, mouse Sertoli (TM4) cells, rat hepatoma (RL12) cells, and human-Chinese hamster ovary (CHO-K1) cells. For 1-NP, a dose range of 1 0 - 2 0 / t g / m l was required to achieve comparable results for the respective cell lines. Only the RL12 and CHO-K1 cells showed recovery of normal DNA synthesis when TNF or 1-NP was removed from the medium. The other cell types showed little or no recovery up to 42 h after removal of the nitroarene. In exclusively studying TNF, the induction of sister-chromatid exchanges (SCEs) and a delay in cell cycle as monitored by harlequin chromosomes, were observed at a concentration range of 0.003-0.2 /xg/ml in PAl, TM4, and RL12 cells. In CHO-K1 cells, TNF at 0.001-1/~g/ml was clearly mutagenic at the hprt locus.
Nitroarenes are common environmental contaminants that exhibit genotoxic activity (Rosenkranz and Mermelstein, 1983, 1985; Tokiwa and Ohnishi, 1986). These compounds, which have been found in photocopier toners (Rosenkranz et al., 1980), diesel exhaust (Salmeen et al., 1982), urban air particulates (Tokiwa et al., 1983) and coal fly ash (Wei et al., 1982), arise from atmo-
Correspondence: Dr. H. Rodriguez,BiologyDepartment, Boston University,2 CummingtonSt., Boston,MA 02215 (U.S.A.).
spheric reactions of polycyclic aromatic hydrocarbons (PAHs) with HNO3 and NO: (Hirayama et al., 1983) and from incomplete combustion processes (Yergey et al., 1982). Nitroarenes are potent direct-acting bacterial mutagens (Rosenkranz and Mermelstein, 1983). In bacterial assays, the mutagenicity is dependent upon enzymatic reduction of the nitro moiety (McCoy et al., 1981; Heflich et al., 1985). In Salmonella t y p h i m u r i u m , these compounds are activated to intermediates capable of forming adducts with the cellular DNA (Rosenkranz, 1982).
0165-1218/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)
74 In contrast to the substantial body of literature concerning the mutagenicity of nitroarenes in Salmonella strains (Rosenkranz and Mermelstein, 1983), the reports based on mammalian (rodent and human) cell assays are less clear and consistent. For instance, Howard and Beland (1982) and Beland et al. (1986) reported that transformation by 1-NP of normal human diploid fibroblasts to a state of anchorage-independent cells, as well as cellular invasiveness, occurred only under an argon atmosphere with the inclusion of xanthine oxidase. By contrast, Patton et al. (1986) induced mutagenic and cytotoxic effects in diploid human fibroblasts with 1-NP in the presence of oxygen and absence of xanthine oxidase. In this paper, we have elected to study the potency of 2 nitroarenes, T N F and 1-NP (in the absence of $9 and presence of oxygen), on 4 different mammalian cell fines using 6 different assay systems. It has been shown that trinitro (as opposed to mononitro and dinitro) compounds generally induce the highest levels of mutagenic and genotoxic actions in various bacteria (Rosenkranz and Mermelstein, 1983) and cultured mammalian cells (Li and Dutcher, 1983). Thus, we have chosen to study the effects of T N F on 4 mammalian cells and compare the results to those produced by 1-NP, a weak mammalian nitroarene (Li and Dutcher, 1983). Part of the conflicting data may be due to different incubation conditions, and the use of different tissue or species. In order to examine whether the discrepancies are due to tissue or species differences, the following cell lines were used: a human teratocarcinoma cell (PAl), a mouse Sertoli cell (TM4), a rat hepatocyte cell (RL12) and a human-Chinese hamster ovarian cell (CHO-K1). Each cell type is useful for a unique purpose: PAl cells are a stable human diploid cell line and have been used previously for mutation assays (Huberman et al., 1984), TM4 cells have characteristics that are consistent with those seen in primary cultures of Sertoli cells (Mather, 1980), RL12 cells are known to metabolize a number of PAHs and might be capable of metabolizing nitroarenes (Murison et al., 1984), and CHO-K1 cells are a stable diploid cell line that has been used previously in mutation assays (Waldren et al., 1979). In doing such a compara-
tive study, we hope to resolve some of the conflicting data on the effects of nitroarenes on various rodent and human cells. Materials and methods
Chemicals 2,4,7-Trinitro-9-fluorenone ( T N F ; purity > 98% as detected by HPLC) and 1-nitropyrene (1-NP; purity > 98% as detected by HPLC) were obtained from the Fluka Chemical Company, Ronkonkoma, NY, while 5-bromodeoxyuridine (BrdUrd), colchicine, and 6-thioguanine (TG) were obtained from the Sigma Chemical Company, St. Louis, MO. The nitroarenes and T G were dissolved in dimethyl sulfoxide (DMSO) prior to their dilution into the culture medium. The BrdUrd and colchicine were dissolved in Dulbecco's phosphate buffer solution (PBS). Cells The characteristics of the PAl clonal isolate obtained from a human teratocarcinoma were described previously (Huberman et al., 1984). The mouse Sertofi cell line TM4 was obtained from primary cultures of Sertoli cell-enriched preparations from normal testis of l l - 1 3 - d a y - o l d B a l b / c n u / + mice (Mather, 1980). The rat hepatoma cell line RL12 had been shown previously to metabolize a number of polycyclic aromatic hydrocarbons (Murison et al., 1984). The human-Chinese hamster cell line CHO-K1 was obtained from fusion of human amniotic fluid fibroblasts and the gly-A mutant of the Chinese hamster ovary cell. This cell contains the standard chromosomes of the CHO-K1 cell plus human chromosome 11 (Puck et al., 1971). All cell lines were cultured routinely in a humidified atmosphere (5% CO 2 in 95% air) in D M E M medium supplemented with 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100/~g/ml) (Gibco, Grand Island, NY). In the cytotoxicity and mutagenicity assays where cloning densities were determined, Ham's F12 (Gibco) supplemented with 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 /~g/ml) was used in place of DMEM. Inhibition of normal DNA synthesis assay All cell lines were subcultured with 0.05% trypsin (Gibco) and transferred to 35-ram dishes
75 (3 dishes/dose) at 1 x 106 cells/dish. After 12-24 h, the cells were treated with the desired concentration of T N F or 1-NP dissolved in DMSO (Mallinckrodt) to a final concentration of DMSO not exceeding 0.05%. After 45 min, [Me3H]thymidine ( 1 / t C i / m l ; 60 Ci/mmole; ICN Radiochemicals, Irvine, CA) was added to each dish. After 3 h of exposure, cells were harvested and lysed overnight in 1 N N H 4 O H and 0.2% Triton X-100 (Fisher Scientific, Pittsburgh, PA). A small aliquot (0.1 ml) of the lysate was removed for total D N A content determination by the use of a T K O 100 DNA fluorometer (Hoefer Scientific Instruments). The rest of the lysate was precipitated with cold 10% trichloroacetic acid, 3% hydrochloric acid and filtered onto G F / A glass microfiber filters (Whatman). The filters were oven dried and placed into a liquid scintillation vial that contained 5 ml of Merrit liquid scintillation cocktail (Isolab). The counts per minute were registered on an LS 100 counter (Beckman).
DNA synthesis recovery assay All cell lines were subcultured with 0.05% trypsin and transferred to 35-mm dishes (3 dishes/dose) at 1 x 10 6 cells/dish. After 12-24 h, the cells were treated with the desired concentration of T N F or 1-NP dissolved in DMSO to a final concentration of DMSO not exceeding 0.05%. After 45 rain, the medium was removed, and fresh culture medium was added. After a 21- and 42-h recovery period, [ Me- 3H]thymidine (1/~ Ci/ml; 60 Ci/mmole) was added. After 3 h of exposure, cells were harvested and lysed overnight in 1 N N H 4 O H and 0.2% Triton X-100. A small aliquot of the lysate was removed for total DNA content determination by the use of a T K O 100 DNA fluorometer. The rest of the lysate was precipitated with cold 10% trichloroacetic acid and 3% hydrochloric acid and filtered onto G F / A glass microfiber filters. The filters were oven dried and placed into a liquid scintillation vial that contained 5 ml of Merrit liquid scintillation cocktail. The counts per minute were registered on an LS 100 counter. Cytotoxicity assay The PAl, TM4, and CHO-K1 cells were subcultured with 0.05% trypsin and transferred to
60-mm dishes (3 dishes/dose) at 500 or 600 cells/dish. After 12-24 h, the cells were treated with the desired concentration of T N F or 1-NP dissolved in DMSO. After 45 min, the medium was removed, and fresh culture medium was added. After 7 days of incubation, colonies were counted by fixing the cells with 70% ethanol and staining with 0.1% crystal violet (Sigma).
Sister-chromatid exchange/harlequin chromosome assay The PAl, RL12, and TM4 cells were subcultured with 0.05% trypsin and transferred to 35-mm dishes at 5 X 10 s cells/dish. After 12-24 h, the cells were treated with the desired concentration of T N F dissolved in DMSO. The culture medium added contained 0.2 m M BrdUrd. The cells were grown in the dark for at least 2 cell cycles (approximately 36 h). Colchicine was added at 0.01 m g / m l for the last 2 h of incubation. The cells were harvested, suspended in hypotonic KC1 (0.075 M), and kept at room temperature for 15 rain. The cells were then fixed in at least 3 changes of glacial acetic acid:methanol solution (1:3), and the cell suspension was dropped onto clean glass slides. 2-day-old slides, kept on a 5 5 ° C slide warmer, were then processed for SCE and percent harlequin analysis by a modification of the hot salt Giemsa technique (Korenberg and Freedlender, 1974). A minimum of 15 sets of chromosomes were scored for SCE/harlequin analysis. Mutagenicity assay CHO-K1 cells were subcultured with 0.05% trypsin and transferred to 100-mm dishes at 2 x 106cells/dish. After 12-24 h, the cells were treated with the desired concentration of T N F dissolved in DMSO. After 45 rain, the medium was replaced with fresh culture medium. After a 7-10-day expression period, cells were subcultured onto 60-mm dishes (10 dishes/dose) at 4 X 10 4 cells/dish. The culture medium contained 10 /~M TG. Additionally, cells were subcultured onto 60-mm dishes (3 dishes/dose) at 500 cells/dish for plating efficiency and cytotoxicity determination. After 7 days, the colonies were counted by fixing the cells with 70% ethanol and staining with 0.1% crystal violet.
76 Results
Inhibition of normal DNA synthesis Fig. 1A shows that inhibition of normal D N A synthesis was greatly increased in all cell types. A 2-way analysis of variance indicated that significant differences in inhibition of normal D N A synthesis between the 4 cell types was evident ( p < 0.0001). In all 4 cell types, the final concentration of T N F required to give 50% inhibition of normal DNA synthesis was 5 /~g/ml. Normal D N A synthesis was inhibited greater than 99% in all 4 cell types when T N F was added at a final concentration of 30 # g / m l . Fig. 1B shows that significant differences in inhibition of normal D N A synthesis were observed between the 4 cell types ( p < 0.0001) when exposed to 1-NP. In all 4 cell types, the final concentration of 1-NP required to give 50% inhibition of normal D N A synthesis was 20/~g/ml. At 30 /zg/ml, 1-NP inhibited normal D N A synthesis no greater than 91% in all 4 cell lines. Therefore, a significant difference was observed between 1-NP and T N F in the degree of inhibition of normal D N A synthesis. This indicates that 1-NP was a less potent inhibitor of normal D N A synthesis than T N F under the present conditions.
DNA synthesis recovery Exposure time of the cells to T N F was based on our previous studies, which showed that 45 rain of exposure produced the same amount of inhibition of normal D N A synthesis as did 3 h and 45 min of exposure (see Figs. 1 and 2). Fig. 2A shows that significant differences in inhibition of normal DNA synthesis were observed between the cell types ( p < 0.0001) when allowed to recover from T N F 21 h after being exposed to the nitroarene. When T N F was at a final concentration of 5 /~g/ml (dosage required for 50% inhibition), both the RL12 and CHO-K1 cells showed recovery from the initial inhibition, while the PAl and TM4 cells did not. Also at this dosage, the RL12 cells showed a stimulation in normal D N A synthesis. Similar results were obtained when the 4 cell types were allowed 42 h of recovery (data not shown). Fig. 2B shows the recovery of normal D N A synthesis after exposure to 1-NP. A significant
difference in inhibition of normal D N A synthesis was observed between the 4 cell types ( p < 0.0001). TM4 cells showed a stimulation in normal DNA synthesis when exposed to 1-NP at a final concentration of 0.2/~g/rnl, while the RL12 cells also exhibited such an effect, but at 5 /~g/ml. When 1-NP was at a final concentration of 20 /~g/ml (dosage required for 50% inhibition), all 4 cell lines exhibited recovery from the initial inhibition. When the cells were allowed 42 h of recovery (data not shown), no differences were observed from those seen at 21 h.
Cytotoxicity Fig. 3 shows that a significant difference ( p < 0.0001) in cytotoxic response to T N F was observed between the PAl, TM4, and CHO-K1 cell types. Overall, the CHO-K1 cells were less affected by the cytotoxic effect of T N F than the PAl and TM4 cells. The cytotoxic potency of 1-NP was less than that for T N F when tested on the PAl, TM4 and CHO-K1 cells (Fig. 4). A significant difference ( p < 0.0001) in cytotoxic response to 1-NP was observed between the 3 cell types. Overall, the TM4 cells were less affected by 1-NP, than the PAl and CHO-K1 ceils. To achieve at least a 50% cytotoxic effect for the PAl, TM4, and CHO-K1 ceils, 1-NP must be added at a final concentration of 25 /~g/ml as opposed to 1 /~g/ml as seen for T N F (Fig. 3). This indicates that T N F was 25 times more cytotoxic than 1-NP. At this point, observing that T N F was far more potent than 1-NP, we decided to focus our studies solely on TNF. Thus 1-NP was not tested for SCEs nor mutagenicity.
Sister-chromatid exchange~harlequin chromosomes Table 1 shows that T N F induced SCEs in PAl, TM4, and RL12 cells. A significant correlation was observed between the dosage of T N F and the number of SCEs per chromosome for the 3 cell types ( p < 0.0001). Significant variations in SCEs per chromosome were detected between the 3 cell types ( p < 0.0001). For the PAl, TM4, and RL12 cells, when the final concentration of T N F exceeded 0.2/~g/ml, cell killing (lysis) was observed. The production of harlequin chromosomes may be used to determine the length of a cell cycle. The
77 effects that T N F had on the cell cycle of the PAl, TM4, and RL12 cells are shown in Table 1. Although the cell cycle of all 3 cell types slowed down with increasing dosage of TNF, the cell cycle of the PAl and TM4 cells slowed down to a greater extent than that seen for the RL12 cells. A correlation between cell cycle and dosage was detected ( p < 0.0001) for the 3 cell types. Significant variations in inhibition of cell cycle were observed between the 3 cell types ( p < 0.0001).
100
10
Mutagenicity
tn cD .c
E < Z
0.1
f
20C
i
|
i
i
i
tx
i
I-NP
Fig. 5 shows the dose-response relationship between mutagen concentrations and mutation frequencies at the hprt locus. Although the frequency of spontaneous mutants for the C H O K1 cells was 12 m u t a n t s / 1 0 5 survivors, the mutant frequency due to T N F at a dosage of 0.001/~g/ml, was 49 mutants/105 survivors. When T N F was at a final concentration of 0.1 # g / m l , the mutant frequency was 80 mutants/105 survivors, and at 1 /~g/ml, T N F induced a mutant frequency of 168 mutants/105 survivors. These data indicate that T N F produced a 4-14-fold increase in mutant frequency over the control.
r
Discussion &
I(X:
0.2
I 5
I 10
I 15 Dose
I 20
I 25
I 30
(,ug / m l )
Fig. 1. (A) DNA synthesis in PAl ([2), TM4 (zx),RL12 (©) and CHO-K1 (I) calls exposed to TNF. The data are presented as the means of triplicates compared to controls without TNF. (B) DNA synthesis in PAl (t3), TM4 (zx), RL12 (o) and CHO-K1 (I) cells exposed to 1-NP. The data are presented as the means of triplicates compared to controls without 1-NP.
In this study, we have shown that T N F was more potent than 1-NP as an inhibitor of normal D N A synthesis. All cell types showed maximal inhibition of normal D N A synthesis in the presence of T N F , while with 1-NP, all 4 cell types showed much less inhibition. Since the experiments were performed in the absence of $9, all 4 cell types must be capable of activating the nitroarenes. In a similar study by Li and Dutcher (1983) using C H O cells, they also reported that trinitro compounds such as 1,3,6-TNP are more potent than 1-NP, as seen by higher levels of mutagenic and genotoxic effects. Both the T M 4 and RL12 cells showed an increase in D N A synthesis over the control rate when exposed to 1-NP at a low concentration of 0.2 /xg/ml. A plausible explanation for such a response might be that this stimulus may be due to D N A repair. Since the activation of nitroarenes by m a m m a l i a n cells forms intermediates capable of forming adducts with the cellular D N A (Patton
78
100 ~
et al., 1986), m a y b e in this study, the cells u n d e r went some form of D N A repair at the low dosages of 1-NP. This would thereby a c c o u n t for the in-
Cytotoxicity
T.
200
TNFA
O 0.01
0.1 Doseof TNF ( }Jg/rnl)
1
Fig. 3. Sur4val of P A l (rn), T M 4 (z~) and C H O - K I ( I ) cells
exposed to TNF. The data are presented as the means of triplicates compared to controls without TNF. Cloning efficiencies were as follows: PAl, 9%; TM4, 21% and CHO-K1, 91%.
10 '
~ ~ 0
[]
in
2OO
1-NP
Q
B
o
°
crease in D N A synthesis over the controls without 1-NP at the low doses. Also, even though it was with a different cell type, 1-NP has b e e n reported to simulate repair synthesis in freshly explanted h u m a n b r o n c h u s epithelial cells (Kawachi, 1982). It is o b v i o u s that studies o n D N A repair m u s t be p e r f o r m e d o n these 4 cell types so as to resolve whether a repair m e c h a n i s m is present or not. W h e n the cells were allowed u p to 42 h to recover from exposure to T N F , only the RL12 a n d C H O - K 1 cells showed some recovery, while the P A l a n d T M 4 cells showed n o recovery within this time period. W h e n the cells were allowed up to 42 h to recover from exposure to 1-NP, the RL12 a n d C H O - K 1 cells showed a greater recovery t h a n the P A l a n d T M 4 cells. This was evident w h e n 1-NP was a d m i n i s t e r e d at 20 a n d 30 / s g / m l . Also, the T M 4 a n d RL12 cells showed this increase i n p e r c e n t D N A synthesis over the con-
10
1
0.2
'
5
o
1
i
i
15 20 Dose (#g/rnl)
21
5
i
30
Fig. 2. (A) DNA synthesis in PAl (C3),TM4 (zx), RL12 (o) and CHO-K1 (11) cells exposed to TNF for 45 min and then allowed 21 h to recover. The data are presented as the means of triplicates compared to controls without TNF. (B) DNA synthesis in PAl (rq), TM4 (zx), RL12 (o) and CHO-K1 (11) cells exposed to 1-NP for 45 rain and then allowed 21 h to recover. The data are presented as the means of triplicates compared to controls without 1-NP.
79
2°°I
Cytotoxicity
TABLE 1 SCEs P E R C H R O M O S O M E CELLS EXPOSED T O T N F
IN PAl, T M 4 A N D RL12
The data are presented as the m e a n ± S.D.; n = 20, except for RL12 where n =15. Also, harlequin chromosomes in PAl, T M 4 and RL12 cells exposed to TNF. Dose of TNF
100
SCEs/chromosome PAl
TM4
% harlequin RL12
PAl
T M 4 RL12
(t,g/w])
-5 .> >
b
(/3 10
1/
0.2
I
5
I
10
'
'
2i
15 20 5 Dose o f I - NP ( ~g / ml )
30
control
0.26+0.1 0.15+0.1 0.205-0.1 100
100
100
0.001 0.003
0.365-0.1 0.34+0.1 0.54-1-0.1 0.42+0.1 0.39+0.1
88.2 77.6
98.6 72.8
91.3
0.01 0.03
0.71+0.1 0.58+0.1 0.495-0.1 1.09+0.1 0.99+0.1 0.78+0.1
70.6 64.7
64.3 57.1
84.8 73.9
0.1 0.3
1.23+0.3 1.22+0.2 1.055-0.3 1.33+0.1 1.45+0.2 1.28+0.2
57.6 50.6
50.0 42.8
63.0 57.6
oxidase. When the TM4 cells were exposed to 1-NP at 0.2-15 # g / m l , cell growth was stimulated above control levels. To explain this stimulation in cell division, future studies on D N A synthesis/cell division need to be performed. Exposure of the cells to T N F proved to be much more cytotoxic than 1-NP. T N F was shown to be 25 times more cytotoxic than 1-NP. CHO-K1 cells were more
Fig. 4. Survival of P A l (D), T M 4 (zx) and CHO-K1 (I) cells exposed to 1-NP. The data are presented as the means of triplicates compared to controls without 1-NP. Cloning efficiencies were as follows: PAl, 17%; TM4, 13% and CHO-K1, 77%.
180 160
140
trol rate when exposed to 1-NP at a concentration range from 0.2 to 20 # g / m l . It has been shown that 1-NP is cytotoxic to diploid human fibroblasts (Patton et al., 1986; Beland et al., 1986). In this study, 1-NP was also cytotoxic to the PAl, TM4, and CHO-K1 cells. These results were obtained in the presence of oxygen and absence of xanthine oxidase. This was contrary to what has been reported previously by Howard and Beland (1982) and Beland et al. (1986) who reported that an oxygen-free atmosphere with the inclusion of xanthine oxidase was required to activate nitroarenes. By contrast, our results are similar to the results obtained by Patton et al. (1986) who were able to induce cytotoxic effects in diploid human fibroblasts with 1-NP in the presence of oxygen and absence of xanthine
LnO 120
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80
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I
0.01 01.1 Dose of TNF ( }Jg/rnl )
110
Fig. 5. D o s e - r e s p o n s e relationship between mutagen concentrations and m u t a n t frequencies at the hprt locus in CHOK1 ( I ) cells. The cloning efficiency of the CHO-K1 cells at the time of selection (7-10-day expression period) was 65 %.
80 resistant to the c y t o t o x i c effect of T N F t h a n were the P A l a n d T M 4 cells. Both the RL12 a n d P A l cells have b e e n shown previously to give rise to SCEs when e x p o s e d to P A H s ( M u r i s o n et al., 1984). I n this study, the P A l , TM4, a n d RL12 cells were e x p o s e d exclusively to T N F a n d then processed for S C E a n a l y sis. A l t h o u g h all 3 cell types showed an increase in SCEs when exposed to T N F , differences in SCEs p e r c h r o m o s o m e were detected b e t w e e n the 3 cell types. Ishidate a n d O d a s h i m a (1977) have shown that carcinogens have the p o t e n t i a l to m a r k e d l y i n h i b i t cell growth. A l t h o u g h the cell cycle of all 3 cell types ( P A l , TM4, a n d RL12) were greatly inhibited b y e x p o s u r e to T N F , the cell cycle of the RL12 cells were slowed to a lesser extent t h a n that observed for the P A l a n d T M 4 cells. In this study, we have shown that T N F was a p o t e n t mutagen, a n d this was i n d i c a t e d b y the m u t a n t frequency p e r g g of mutagen. T h e results o b t a i n e d here were o b t a i n e d in the presence of oxygen a n d absence of x a n t h i n e oxidase. This was similar to the results o b t a i n e d b y P a t t o n et al. (1986) who i n d u c e d m u t a g e n i c effects in d i p l o i d h u m a n fibroblasts with 1-NP in the presence of oxygen a n d absence of x a n t h i n e oxidase. Thus, n o special i n c u b a t i o n c o n d i t i o n s were r e q u i r e d as app o s e d to that r e p o r t e d b y H o w a r d a n d B e l a n d (1982) a n d B e l a n d et al. (1986). I n conclusion, we have d e m o n s t r a t e d that T N F was significantly m o r e genotoxic t h a n 1 - N P in all 4 cell lines a n d significantly m u t a g e n i c to C H O - K 1 cells. Also, no special i n c u b a t i o n c o n d i t i o n s (oxygen-free a t m o s p h e r e with the inclusion of xanthine oxidase) were r e q u i r e d to activate the nitroarenes. A l l 4 cell lines, r e p r e s e n t a t i v e of 4 species a n d 3 different tissues, were c a p a b l e of activating b o t h nitroarenes, a n d showed similar qualitative responses b u t different q u a n t i t a t i v e responses to these 2 nitroarenes. F u r t h e r d e t e r m i n a tion of the n i t r o r e d u c t a s e levels in each cell line w o u l d p r o v i d e useful i n f o r m a t i o n for u n d e r s t a n d ing the basis of these results.
Acknowledgement This w o r k was s u p p o r t e d b y a g r a n t f r o m the N a t i o n a l I n s t i t u t e of E n v i r o n m e n t a l H e a l t h to Dr. G e r a l d L. Murison.
References Beland, F.A., M. Ribovich, P.C. Howard, R.H. Heflich, P. Kurian and G.E. Milo (1986) Cytotoxicity, cellular transformation and DNA adducts in normal human diploid fibroblasts exposed to 1-nitrosopyrene, a reduced derivative of the environmental contaminant, 1-nitropyrene, Carcinogenesis, 7, 1279-1283. Heflich, R.H., P.C. Howard and F.A. Beland (1985) 1-Nitrosopyrene: an intermediate in the metabolic activation of 1-nitropyrene to a mutagen in Salmonella typhimurium TA1538, Mutation Res., 149, 25-32. Hirayama, T., M. Nohara, T. Ando, M. Tanaka, K. Nagano and S. Fukui (1983) Mutagenicity of coal-pyrolyzed products and their photochemical reaction products with nitrogen oxides in Salmonella typhimurium TA98 and TA100, Mutation Res., 122, 273-278. Howard, P.C., and F.A. Beland (1982) Xanthine oxidase catalyzed binding of 1-nitropyrene to DNA, Biochem. Biophys. Res. Cornrnun., 104, 727-732. Huberman, E., C.K. McKeown, C.A. Jones and S. Murao (1984) Induction of mutations by chemical agents at the hypoxanthine guanine phosphoribosyltransferase locus in human epithelial teratoma cells, Mutation Res., 130, 127-137. lshidate, M., and S. Odashima (1977) Chromosome tests with 134 compounds on Chinese hamster cells in vitro - - A screening for chemical carcinogens, Mutation Res., 48, 337-354. Kawachi, T. (1982) Mutagenicity and carcinogenicity of nitropyrenes, in: D.E. Rickert (Ed.), The Toxicity of Nitroaromatic Compounds, Hemisphere Press, New York. Korenberg, J.R., and E.F. Freedlender (1974) Giemsa technique for the detection of sister chromatid exchanges, Chromosoma, 48, 355-360. Li, A.P., and J.S. Dutcher (1983) Mutagenicity of mono-, di-, and tri-nitropyrenes in Chinese hamster ovary cells, Mutation Res., 119, 387-392. Mather, J.P. (1980) Establishment and characterization of 2 distinct mouse testicular epithelial-cell lines, Biol. Reprod., 23, 243-252. McCoy, E.C., H.S. Rosenkranz and R. Mermelstein (1981) Evidence of the existence of a family of bacterial nitroreductases capable of activating nitrated polycyclics to mutagens, Environ. Mutagen, 3, 421-427. Murison, G.L., S.D. Hall and A.M. Lee (1984) Metabolic activation of procarcinogens to genotoxic products in cultured rat liver cells, Exp. Cell. Biol., 52, 355-360. Patton, J.D., V.M. Maher and J.J. McCormick (1986) Cytotoxic and mutagenic effects of 1-nitropyrene and 1-nitrosopyrene in diploid human fibroblasts, Carcinogenesis, 7, 89-93. Puck, T.T., P. Wuthier, C. Jones and F.T. Koa (1971) Genetics of somatic mammalian cells: lethal antigens as genetic markers for study of human linkage groups, Proc. Natl. Acad. Sci. (U.S.A.), 68, 3102-3106. Rosenkranz, H.S. (1982) Direct-acting mutagens in diesel exhausts: magnitude of the problem, Mutation Res., 101, 1-10.
81 Rosenkranz, H.S., and R. Mermelstein (1983) Mutagenicity and genotoxicity of nitroarenes: all nitro-containing chemicals were not created equal, Mutation Res., 114, 217-267. Rosenkranz, H.S., and R. Mermelstein (1985) The genotoxicity, metabolism and carcinogenicity of nitrated polycychc aromatic hydrocarbons, J. Environ. Sci. Health, 2, 221-272. Rosenkranz, H.S., E.C. McCoy, D.R. Sanders, M. Butler, D.K. Kiriazides and R. Mermelstein (1980) Nitropyrenes: isolation, identification, and reduction of mutagenic impurities in carbon black and toners, Science, 209, 1039-1043. Salmeen, I., A.M. Durisin, T.J. Prater, T. Riley and D. Schuetzle (1982) Contribution of 1-nitropyrene to direct-acting Ames assay mutagenicities of diesel particulate extracts, Mutation Res., 104, 17-23. Tokiwa, H., and Y. Ohnishi (1986) Mutagenicity and carcino-
genicity of nitroarenes and their sources in the environment, CRC C. R. Toxicol., 17, 23-60. Tokiwa, H., S. Kitamori, R. Nakagawa, K. Horikawa and L. Matamala (1983) Demonstration of a powerful mutagenic dinitropyrene in airborne particulate matter, Mutation Res., 121, 107-116. Waldren, C., C. Jones and T.T. Puck (1979) Measurement of mutagenesis in mammalian cells, Proc. Natl. Acad. Sci. (U.S.A.), 76, 1358-1362. Wei, C., O.G. Raabe and L.S. Rosenblatt (1982) Microbial detection of mutagenic nitro-organic compounds in filtrates of coal fly ash, Environ. Mutagen., 4, 249-258. Yergey, J.A., T.N. Risby and S.S. Lestz (1982) Chemical characterization of organic adsorbates on diesel particulate matter, Anal. Chem., 54, 354-357.