- Email: [email protected]
The genetic effects of the cytostatic drug TS160 on Chinese hamster fibroblasts in vitro
Mutation Research, 116 (1983) 431-440 Elsevier Biomedical Press
431
The genetic effects of the cytostatic drug TSi60 on Chinese hamster fibroblasts in vitro D. Slmne~ovh, M. Du~inskit, E. B u d a y o v / l a n d A. G a b e l o v h Department of Mutagenesis, Cancer Research Institute, Slovak Academy of Sciences, Ceskoslovenskej armddy 21, 812 32 BratiMava (Czechoslovakia)
(Received 26 Janu~iry1982) (Revision received 29 July 1982) (Accepted 6 August 1982)
Summaw The cytostatic agent TSt60 inhibited DNA synthesis in V79 cells while the course of RNA synthesis and protein synthesis were not changed significantly, DNA inhibition was in direct correlation with the inhibition of growth and reduction of colony-forming ability in the treated cells, The study of the mutagenic consequences of single or repeated TSl60 treatments showed that TSI60 had a significant mutagenic activity on Chinese hamster V79 cells. In the study of mutagenic effects of TS~60, we used the method of repeated treatment of surviving cell fractions with this compound. This method is suitable for those mutagens (e.g. weak mutagens) whose mutagenic activity on mammalian cells in vitro might escape attention after application of a single dose. The possible causes of mutagenic effects of TSI60 are discussed.
The biological effects of yperite and its derivatives are comparable to the effects of X-rays. For these similarities, nitrogen mustard has been used as the first chemical compound in the chemotherapy of cancer. Nitrogen mustard (HN 2), however, has relatively high toxic effects as well; therefore some other derivatives of this compound were sought for clinical practice. In 1947, TSI6o (tris-fl-chloroethylamine= HN3) was synthesized. Chemically, TSl60 is similar to HN2; TSI6o comprises three, HN 2 two, chloroethyl groups. Both nitrogen mustard and TS16o belong to the group of alkylating agents that react with biologically important macromolecules - mainly DNA -- by adding alkyl groups to these molecules. The conflicting results of mutagenicity experiments with HN 2 in mammalian cells (Anderson and Fox, 1974; Suter et al., 1980) led us to study the genetic effects of TS~60 on Chinese hamster V79 fibroblasts. Our preliminary results were in good correspondence with the results obtained by Suter et al. (1980). We did not find any 0165-1218/83/0000-0000/$03.00 © Elsevier Biomedical Press
432 mutagenic activity of YS]6 0 at higher concentrations (unpublished results) despite the DNA-inhibiting effects of this substance on human fibroblasts EUE (Slamefiovfi et al., 1980). The absence of mutagenic activity was surprising because both HN 2 and TS]6 0 a r e known as directly acting carcinogens (Griffin et al., 1951; Heston, 1953; Shimkin et al., 1966; S~¢kora et al., 1981). A detailed study of TS~60-induced mutagenicity after the application of a wide range of concentrations and the mutagenicity study of repeated treatment have convinced us, however, about the significant mutagenic effects of TSj6 0 o n V79 cells.
Materials and methods
Cell line. We used near-diploid V79 hamster cells (obtained from Dr. A. Abbondandolo, Laboratory of Mutagenesis, Pisa, Italy). The cells were cultivated in Eagle's minimal essential medium (Gibco), supplemented with 10% calf serum, 2.5% foetal calf serum, a mixture of non-essential amino acids, 0.1 g sodium pyruvate per 1000 ml, penicillin (200 U / m l ) , streptomycin (100 ffg/ml) and kanamycin (100 ffg/ml) in a humidified 5% CO 2 atmosphere at 37°C in glass petri dishes. The cells were checked under an electron microscope for the presence of PPLO; no contamination was noted. Chemical. TS16 0 (pure compound) was purchased from L6~iva, Prague. The substance was diluted in the medium immediately before application. Treatment of cells. About 2 x 10 6 exponentially growing V79 cells (3.5 x l 0 4 cells/cm 2) were treated with 0-10 /~g TSi60/ml. (TSl6 0 w a s diluted in complete medium, in a medium without serum or in PBS buffer.) Then the cells were washed, resuspended by means of 0.025% trypsin in 0.02% ethylenediaminotetraacetic acid (EDTA) and tested for their growth activity, plating efficiency, course of macromolecular syntheses and for the presence of 6-thioguanine-resistant (6-TG r) mutants. Growth activity. The treated cells were plated on a series of petri dishes, diameter 5 cm (4 x 10 4 cells per dish) and incubated at 37°C. At 24-h intervals the cells were removed from individual petri dishes and the numbers of cells/dish were counted. Plating efficiency. After treatment the cells were appropriately diluted and plated on 3-5 petri dishes, diameter 5 cm, in an amount of 2 x 102-5 x 10Z/dish. Macromolecular syntheses. Both treated and control cells were grown in a medium containing either [t4C]thymidine (Tdr) (1 ffCi/ml) or [14C]uridine (Urd) (1 /~Ci/ml) or [14C]-L-leucine (0.2 /LCi/ml). At various intervals after treatment, the labelling was terminated by rapid sucking off of the medium, rinsing of the cells in an SSC buffer (0.15 M sodium chloride, 0.015 M sodium citrate) and by adding 3 ml 5% trichloroacetic acid (TCA) cooled to 0°C. The precipitated cells were stored for 24 h at 5°C; the cell material was then removed from the glass with a silicone scraper. The cells were resuspended, and 2 x 1-ml aliquots were filtered through a SYNPOR 6-membrane filter. The membranes were washed with 5% TCA, 3-timeso distilled water and 96% ethanol. The membranes were dried and their radioactivity was measured on a liquid scintillation counter Packard Tri Carb.
433
Mutagenicity assay. The assay for the detection of 6-TG r mutations was carried out as described previously (Slamei~ovh and Gabelovh, 1980). Essentially, V79 cells were kept in exponential growth for 6-11 days after treatment with TSi60; then they were plated on 5 dishes, diameter 10 cm (5 × 105 cells/dish). After the cells had attached, 6-thioguanine (5/~g/ml) was added and 2 × 102-1 × 103 cells were plated on 5 petri dishes to determine plating efficiency. The genetic effects of 4 subsequent treatments at different concentrations of T516o were studied in experiments where the second, the third and the fourth treatments were performed on days 8, 16 and 24 following the first treatment. The mutagenic response was scored after the first, the second and the fourth treatments with TS160. Results and discussion
Like nitrogen or sulphur mustard or some other chemicals, TS160 belongs to the group of directly acting chemical compounds, the so-called primary carcinogens. These substances attach to nucleic acids, usually resulting in alkylation or acylation of the bases. As an introduction to the investigation of the mutagenic activity of TSl6 o on V79 cells we studied the course of DNA and RNA syntheses during several hours after treatment with TSl6o. Fig. 1 shows that TSl60 exhibited strong DNA-inhibiting effects dependent on the concentration used. On the other hand, a slight inhibition of RNA synthesis was observed only 3 h after treatment with TSI60 (Fig. 2). These marked differences in negative effects of TS~60 on the synthesis of D N A and RNA may be explained by the numerous modifications of the primary structure of DNA and RNA (O'Connor, 1981). With respect to the significant DNA-inhibiting effects of this alkylating agent, it may be important that the N-glycosidic bond is unstable in the 3- and 7-alkylpurine nucleotides of DNA, so that a free base is readily lost on depurination. On the contrary, in RNA this bond is stable under physiological conditions (Lawley and Brookes, 1963). The results obtained from the studies on the course of protein synthesis in the treated cells show that T5160 does not inhibit total synthesis (Fig. 3). As we found earlier (Slamefiovh et al., 1980), similar effects of this substance on the synthesis of D N A and proteins had exhibited the human EUE fibroblasts. To study the mutagenic effects of TSl60 at different levels of the surviving fraction of V79 cells, it was necessary to determine the cytotoxicity of this compound. We did so either on the basis of the growth activity in the treated cells (Fig. 4) or by counting the percentage of colony-forming ability (Fig. 5). The inhibition of growth (Fig. 4) in the treated V79 cells is in good correlation with the DNA inhibition (Fig. 1). It is obvious that the cytotoxicity of this compound is dependent on its toxic activity on DNA. From the curves of growth activity (Fig. 4) it was possible to choose convenient concentrations of TSl6o for testing the plating efficiency (colonyforming ability), which is a more exact criterion for determination of the surviving fraction. The colony-forming ability was studied after the cells had been treated with TSI60 in different conditions. Treatment in the complete medium was less toxic than that in a medium without serum or in the buffer (Fig. 5). For the mutagenicity studies all types of treatment were used. From Fig. 6 it is apparent that the
434
15
e•
10
5
0 o
rk
'
1~o
' 3o'o
'
rain of post-TS16 O-treatment Fig. 1.
25I=
Q.
o
20-
x
15-
10-
5"
0 0
60
120
180
240
300
min of post-TS160-treotment Fig. 2.
435
15" E
e~ o N o x
10-
5-
0
b
6
I
120
I
I
I
240
rnin of post-TS160-treatrnent Fig. 3.
Figs. 1, 2 and 3. The course of DNA synthesis (Fig. 1), RNA synthesis (Fig. 2) and protein synthesis (Fig. 3) in control V79 cells and in the cells treated with TSI60 for 60 rain. O, 0.5/~g/ml; t,, 1/~g/ml; A, 3 #g/ml; I, 5 #g/ml; D, 10 #g/ml; o, control. mutagenic effect is not dependent on the manner of treatment, but is in close relationship with the level of survival. The frequency of 6-TG r mutations increased in the span of survival from 90 to 10%; however, when the surviving fraction fell below 10%, the cytotoxic effects of TS16o covered the mutagenic effects and the frequency of 6-TG r mutations gradually decreased. When the survival was 1% or less we could not find any induced 6-TG r mutations in the population of surviving cells. The rather low mutagenic effects of TS160 found in V79 cells after a single treatment (Fig. 6) led us to test the effect of repeated treatment with this substance. We found that 4 treatments with TS~60, at 8-day intervals, increased the sensitivity of V79 cells to this substance only partly, while the mutagenic effects of the 4 treatments had a roughly additive character (Table 1). It is interesting to compare this result with those obtained from experiments with chronic treatment of rats with N, N-dimethylnitrosamine ( D M N ) (Montesano et al., 1980), where the livers of treated animals augmented their ability to repair O6-meth ylguanine (i.e., D N A damage), which is considered, in respect of mutagenic effects, to be the most significant. Thus, a decreased rather than an increased mutagenic effect following repeated doses of the alkylating agent could be expected. However, as discussed below, production of O6-alkylguanine is not the only D N A damage caused by TSI6o. Similarly, the highly specific properties of liver cells can hardly be generalized.
106
1
days after
treatment
Fig. 4. Growth activity of V79 cells after 60 min incubation with various concentrations of TSI60. O, 0.5 /.tg/ml; A, 1 /~g/ml; &, 3 /~g/ml; I , 5 /~g/ml; D, 10 btg/ml; e, control.
100 -
~,,,,,
0"'-
-o..p 0
o
, ,,
o'" "0"'-% °
o
0
\
10. It,. O
\ \ \, tn t
.-e
-0
\
'~x t \ I
\ \ t
I
I I
0
Y!
6s
I 4ag TS160/m[
Fig. 5. Colony-forming ability of V79 cells after 60 rain incubation with various concentrations of TS:6 o in complete medium (O), and after 30 min of incubation in a medium without serum (zx) and in PBS buffer
(D).
437
10
o =8
m
~7 '%
bi
~
?
? i
'
6
8
4
z~
3 I
I1~
100
|
|
I
I
I
|11
10
i1!
1
i
o/. OF
|
i
iii
1 SURVIVORS
i
i~
iimiii
0.1
Fig. 6. Frequency of induced 6-TG r mutations in V79 cells treated with various concentrations of TSt60 in complete medium (60 min, ©), in medium without serum (30 min, zx) and in PBS buffer (30 min, n). The individual points represent mean values obtained from 3 different Expts. For determination of 1 point 2.5 × 10 6 cells from 5 petri dishes were tested.
'
The resistance of several spontaneous 6-TG r cell lines t o TS]60 was observed in further experiments to eliminate the possibility that TSI60 had not induced, but had selected, 6-TG r mutations. Our results showed that spontaneous 6-TG r cells (isolated from V79 cells) can be characterized by the same or lower resistance to TS~60 than the V79 cells. The selection of spontaneous 6-TG r mutations by TSI60 is therefore not probable. How can we explain the mutagenicity of TS~60? Alkylating agents usually attach to D N A in the N-7 position of guanine. These modifications of guanine had therefore been considered to be the source of mutations. It was established, however (Lawley, 1974), that, with respect to mutagenicity, the alkylation of guanine in the 0 - 6 position was more important because it changed the pairing capacity of guanine. F r o m this aspect, alkylation in further positions of D N A bases (0-4 thymine; N-3 guanine; N-1 and N-7 adenine; N-3 cytosine and N-3 and N-4 thymine) must be important (Auerbach, 1976). It is necessary to emphasize that the induction of mutations is only a rare event, so that only few cases of alkylation result in mutation. The secondary effects of alkylation have to be considered as the other alternative for the origin of mutations in alkylated DNA. As mentioned above, N-glycosidic bonds are unstable in guanines alkylated in N-3 and N-7 positions. This situation m a y result in creation of apurinic sites or further in single-strand breaks of DNA. After alkylation of D N A by bifunctional or polyfunctional agents ( H N 2, T S l 6 0 ) ,
0 1 1.5 1.75 2 2.25 2.5 2.75 3
S e c o n d treatment
O 1 1.5 1.75 2 2.25 2.5 2.75 3
First treatment
TSI6o (/~g/ml)
81.8 63.2 68.5 40.4 44 40.1 30.6 29.9 20.5
96 62.7 55.8 46.3 42.5 25.8 28.9 25.2 25
PE (AT)
74.6 59.8 82.3 73.4 91.1 78.8 95.9 68.7 74
0.164+0.1313 1.070 -+ 1.2641 1.798 + 0.6262 1.089 + 0.3337 1.229+0.1075 1.218 + 0.6663 0.959 + 0.5374 1.647 -+0.9497 8.702 + 0.8614
1 6.52 10.69 6.64 7.49 7.42 5.84 10.05 53.06
R
72.7 83.1 86.9 61.0 76.9 99.9 71.0 68.8 105.6
PE (%)
M -+ o
89.6 92.1 80.8 89.5 73.2 89.3 70.2 85.7 76.5
PE (%)
1 4.68 3.29 13.09 2.02 3.69 2.92 9.28 21.97 11 days
0.154±0.1244 0.722±0.5476 0.508 -+0.3218 2.017 -+0.5814 0.312-+0.2846 0.569 -+0.1163 0.450-+0.3820 1.430-+0.3103 3.389 -+0.8228
8 days
77.6 99.6 78.6 81.3 102.2 84.3 87.6 69.9 86.3
PE (%)
R
PE (%)
M± o
9 days
7 days
Expression time
OCCURRENCE OF 6-TG r MUTATIONS IN V79 CELLS AFTER 1, 2 A N D 4 TREATMENTS WITH TSI6o
TABLE 1
1
12.29 6.97 26.03
3.380 _+0.9257 1.910 +0.6527 7.154 +0.5213
1
7.96 10.56 9.29 2.7 1.5 3.2 7.07 10.28
2.27 4.74 11.19 2.08
-+0.2255 +0.9613 -+0.9981 +0.7915 -+0.5080 -+0.2194 -+0.4029 ± 0.5622 -+0.6186
_+0.2460 + 0.2454 _+0.4153 _+ 1.2494 _+0.3176
0.275 0.625 1.304 3.079 0.572
M_.+o
0.178 1.52 1.881 1.653 0.491 0.268 0.569 1.259 1.830
M-+o
O~
17.2 15.7 13.5 lO
2.25 2.5 2.75 3
. 74.1 89.1 92.6
90.8 79.8 61.6 73.9 80.3 .
. 1.942 ± 0.6378 6.690 ± 0.6912 20.605±2.2782
0.149 + 0.1837 1.203 _+0.4010 3.051 + 0 . 7 8 4 6 1.510 +_0.4220 2.760+0.2672 . 13.03 44.91 138.25
1 8.07 20.47 10.13 18.52 . 93 51.4 64.2
83.9 58 82.9 91.5 71.7 24.534 ± 1.3618
0.2010+0.1786 1.655 _+0.5500 3.570 + 1.0393 3.730 _____1.0509
M± o
x i is the sum of n u m b e r s of 6 - T G r m u t a n t s per 100000 cells in i n d i v i d u a l samples, p is the m e a n of these values, n is their number. R , ratio of i n d u c e d to s p o n t a n e o u s m u t a t i o n s .
P E (AT), p l a t i n g efficiency i m m e d i a t e l y after treatment. M ± o, average n u m b e r s of 6 - T G r m u t a n t s per 100000 cells + s t a n d a r d d e v i a t i o n o. The s t a n d a r d d e v i a t i o n o was c a l c u l a t e d by the e q u a t i o n
86.6 71.8 42.2 41.7 25
Fourth treatment
0 1 1.5 1.75 2
P E (%)
R
PE (%)
M± o
8 days
6 days
122.03
18.59
1 8.23 17.76
".O
440 often cross-links between the DNA strands (mostly between the guanines in N-7 positions) may be formed. These can be excised spontaneously or enzymatically, l e a v i n g d o u b l e - s t r a n d b r e a k s i n t h e D N A . I t is p o s s i b l e t h a t t h e s e c o n d a r y m u t a g e n i c e f f e c t s o f D N A a l k y l a t i o n r e s i d e i n i r r e g u l a r f i l l i n g u p o f t h e a p u r i n i c sites i n DNA
or in erroneous repair of single- or double-strand breaks in DNA.
Acknowledgements We are greatly indebted solicitous technical help.
t o M r s . A. M o r / t v k o v ~ a n d M r s . H . P l ~ t e n i k o v ~ f o r
References Anderson, D., and M. Fox (1974) The induction of thymine and IUdR resistant variants in P388 mouse lymphoma cells by X-rays, UV and mono- and bi-functional alkylating agents, Mutation Res., 25, 107-122. Auerbach, C. (1976) The chemical mutagens: alkylating agents, in: Chapman and Hall (Eds.), Mutation Research; Problems, Results and Perspectives, A Halsted Press Book, John Wiley, New York, Ch. 16. Griffin, A.C., E.L. Brandt and E.L. Tatum (1951) Induction of tumors with nitrogen mustards, Cancer Res., 11,253-260. Heston, W.E. (1953) Occurrence of tumors in mice injected subcutaneously with sulphur mustard and nitrogen mustard, J. Natl. Cancer Inst., 14, 131-148. Lawley, P.D. (1974) Some chemical aspects of dose-response relationships in alkylation mutagenesis, Mutation Res., 23, 283-295. Lawley, P.D., and P. Brookes (1963) Further studies on the alkylation of nucleic acids and the constituent nucleotides, Biochem. J., 89, 127-138. O'Connor, P.J. (1981) Studies on mechanism of action; Interaction of chemical carcinogens with macromolecules, J. Cancer Res. Clin. Oncol., 99, 167-186. Montesano, R., H. Br/:sil, G. Planche-Martel, G.P. Margison and A.E. Pegg (1980) Effect of chronic treatment of rats with dimethylnitrosamine on the removal of O6-methylguanine from DNA, Cancer Res., 40, 452-458. Shimkin, M.B., J.H. Weisburger, E.K. Weisburger, N. Gubareff and V. Suntzeff (1966) Bioassay of 29 alkylating chemicals by the pulmonary-tumor response in strain A mice, J. Natl. Cancer Inst., 36, 915-935. Slamefiovb., D., and A. Gabelovh (1980) The effects of sodium azide on mammalian cells cultivated in vitro, Mutation Res., 71,253-261. Slamefiovb,, D., A. Gabelovb. and M. Kothajovb. (1980) Notes to application of the DNA synthesis inhibition test in the screening of DNA-damaging factors, Studia Biophys., 78, 3, 165-175. Suter, W., J. Brennand, S. McMillan and M. Fox (1980) Relative mutagenicity of antineoplastic drugs and other alkylating agents in V79 Chinese hamster cells, independence of cytotoxic and mutagenic response, Mutation Res., 73, 171-181. S~'kora, I., V. Vortel, O. Marhan and A. Dynterovh (1981) Carcinogenicity of trichlormethine hydrochloride (ZSl60 Spofa) and morphological damage after its intraamniotic injection, Neoplasma. 28, 5, 565-574.
431
The genetic effects of the cytostatic drug TSi60 on Chinese hamster fibroblasts in vitro D. Slmne~ovh, M. Du~inskit, E. B u d a y o v / l a n d A. G a b e l o v h Department of Mutagenesis, Cancer Research Institute, Slovak Academy of Sciences, Ceskoslovenskej armddy 21, 812 32 BratiMava (Czechoslovakia)
(Received 26 Janu~iry1982) (Revision received 29 July 1982) (Accepted 6 August 1982)
Summaw The cytostatic agent TSt60 inhibited DNA synthesis in V79 cells while the course of RNA synthesis and protein synthesis were not changed significantly, DNA inhibition was in direct correlation with the inhibition of growth and reduction of colony-forming ability in the treated cells, The study of the mutagenic consequences of single or repeated TSl60 treatments showed that TSI60 had a significant mutagenic activity on Chinese hamster V79 cells. In the study of mutagenic effects of TS~60, we used the method of repeated treatment of surviving cell fractions with this compound. This method is suitable for those mutagens (e.g. weak mutagens) whose mutagenic activity on mammalian cells in vitro might escape attention after application of a single dose. The possible causes of mutagenic effects of TSI60 are discussed.
The biological effects of yperite and its derivatives are comparable to the effects of X-rays. For these similarities, nitrogen mustard has been used as the first chemical compound in the chemotherapy of cancer. Nitrogen mustard (HN 2), however, has relatively high toxic effects as well; therefore some other derivatives of this compound were sought for clinical practice. In 1947, TSI6o (tris-fl-chloroethylamine= HN3) was synthesized. Chemically, TSl60 is similar to HN2; TSI6o comprises three, HN 2 two, chloroethyl groups. Both nitrogen mustard and TS16o belong to the group of alkylating agents that react with biologically important macromolecules - mainly DNA -- by adding alkyl groups to these molecules. The conflicting results of mutagenicity experiments with HN 2 in mammalian cells (Anderson and Fox, 1974; Suter et al., 1980) led us to study the genetic effects of TS~60 on Chinese hamster V79 fibroblasts. Our preliminary results were in good correspondence with the results obtained by Suter et al. (1980). We did not find any 0165-1218/83/0000-0000/$03.00 © Elsevier Biomedical Press
432 mutagenic activity of YS]6 0 at higher concentrations (unpublished results) despite the DNA-inhibiting effects of this substance on human fibroblasts EUE (Slamefiovfi et al., 1980). The absence of mutagenic activity was surprising because both HN 2 and TS]6 0 a r e known as directly acting carcinogens (Griffin et al., 1951; Heston, 1953; Shimkin et al., 1966; S~¢kora et al., 1981). A detailed study of TS~60-induced mutagenicity after the application of a wide range of concentrations and the mutagenicity study of repeated treatment have convinced us, however, about the significant mutagenic effects of TSj6 0 o n V79 cells.
Materials and methods
Cell line. We used near-diploid V79 hamster cells (obtained from Dr. A. Abbondandolo, Laboratory of Mutagenesis, Pisa, Italy). The cells were cultivated in Eagle's minimal essential medium (Gibco), supplemented with 10% calf serum, 2.5% foetal calf serum, a mixture of non-essential amino acids, 0.1 g sodium pyruvate per 1000 ml, penicillin (200 U / m l ) , streptomycin (100 ffg/ml) and kanamycin (100 ffg/ml) in a humidified 5% CO 2 atmosphere at 37°C in glass petri dishes. The cells were checked under an electron microscope for the presence of PPLO; no contamination was noted. Chemical. TS16 0 (pure compound) was purchased from L6~iva, Prague. The substance was diluted in the medium immediately before application. Treatment of cells. About 2 x 10 6 exponentially growing V79 cells (3.5 x l 0 4 cells/cm 2) were treated with 0-10 /~g TSi60/ml. (TSl6 0 w a s diluted in complete medium, in a medium without serum or in PBS buffer.) Then the cells were washed, resuspended by means of 0.025% trypsin in 0.02% ethylenediaminotetraacetic acid (EDTA) and tested for their growth activity, plating efficiency, course of macromolecular syntheses and for the presence of 6-thioguanine-resistant (6-TG r) mutants. Growth activity. The treated cells were plated on a series of petri dishes, diameter 5 cm (4 x 10 4 cells per dish) and incubated at 37°C. At 24-h intervals the cells were removed from individual petri dishes and the numbers of cells/dish were counted. Plating efficiency. After treatment the cells were appropriately diluted and plated on 3-5 petri dishes, diameter 5 cm, in an amount of 2 x 102-5 x 10Z/dish. Macromolecular syntheses. Both treated and control cells were grown in a medium containing either [t4C]thymidine (Tdr) (1 ffCi/ml) or [14C]uridine (Urd) (1 /~Ci/ml) or [14C]-L-leucine (0.2 /LCi/ml). At various intervals after treatment, the labelling was terminated by rapid sucking off of the medium, rinsing of the cells in an SSC buffer (0.15 M sodium chloride, 0.015 M sodium citrate) and by adding 3 ml 5% trichloroacetic acid (TCA) cooled to 0°C. The precipitated cells were stored for 24 h at 5°C; the cell material was then removed from the glass with a silicone scraper. The cells were resuspended, and 2 x 1-ml aliquots were filtered through a SYNPOR 6-membrane filter. The membranes were washed with 5% TCA, 3-timeso distilled water and 96% ethanol. The membranes were dried and their radioactivity was measured on a liquid scintillation counter Packard Tri Carb.
433
Mutagenicity assay. The assay for the detection of 6-TG r mutations was carried out as described previously (Slamei~ovh and Gabelovh, 1980). Essentially, V79 cells were kept in exponential growth for 6-11 days after treatment with TSi60; then they were plated on 5 dishes, diameter 10 cm (5 × 105 cells/dish). After the cells had attached, 6-thioguanine (5/~g/ml) was added and 2 × 102-1 × 103 cells were plated on 5 petri dishes to determine plating efficiency. The genetic effects of 4 subsequent treatments at different concentrations of T516o were studied in experiments where the second, the third and the fourth treatments were performed on days 8, 16 and 24 following the first treatment. The mutagenic response was scored after the first, the second and the fourth treatments with TS160. Results and discussion
Like nitrogen or sulphur mustard or some other chemicals, TS160 belongs to the group of directly acting chemical compounds, the so-called primary carcinogens. These substances attach to nucleic acids, usually resulting in alkylation or acylation of the bases. As an introduction to the investigation of the mutagenic activity of TSl6 o on V79 cells we studied the course of DNA and RNA syntheses during several hours after treatment with TSl6o. Fig. 1 shows that TSl60 exhibited strong DNA-inhibiting effects dependent on the concentration used. On the other hand, a slight inhibition of RNA synthesis was observed only 3 h after treatment with TSI60 (Fig. 2). These marked differences in negative effects of TS~60 on the synthesis of D N A and RNA may be explained by the numerous modifications of the primary structure of DNA and RNA (O'Connor, 1981). With respect to the significant DNA-inhibiting effects of this alkylating agent, it may be important that the N-glycosidic bond is unstable in the 3- and 7-alkylpurine nucleotides of DNA, so that a free base is readily lost on depurination. On the contrary, in RNA this bond is stable under physiological conditions (Lawley and Brookes, 1963). The results obtained from the studies on the course of protein synthesis in the treated cells show that T5160 does not inhibit total synthesis (Fig. 3). As we found earlier (Slamefiovh et al., 1980), similar effects of this substance on the synthesis of D N A and proteins had exhibited the human EUE fibroblasts. To study the mutagenic effects of TSl60 at different levels of the surviving fraction of V79 cells, it was necessary to determine the cytotoxicity of this compound. We did so either on the basis of the growth activity in the treated cells (Fig. 4) or by counting the percentage of colony-forming ability (Fig. 5). The inhibition of growth (Fig. 4) in the treated V79 cells is in good correlation with the DNA inhibition (Fig. 1). It is obvious that the cytotoxicity of this compound is dependent on its toxic activity on DNA. From the curves of growth activity (Fig. 4) it was possible to choose convenient concentrations of TSl6o for testing the plating efficiency (colonyforming ability), which is a more exact criterion for determination of the surviving fraction. The colony-forming ability was studied after the cells had been treated with TSI60 in different conditions. Treatment in the complete medium was less toxic than that in a medium without serum or in the buffer (Fig. 5). For the mutagenicity studies all types of treatment were used. From Fig. 6 it is apparent that the
434
15
e•
10
5
0 o
rk
'
1~o
' 3o'o
'
rain of post-TS16 O-treatment Fig. 1.
25I=
Q.
o
20-
x
15-
10-
5"
0 0
60
120
180
240
300
min of post-TS160-treotment Fig. 2.
435
15" E
e~ o N o x
10-
5-
0
b
6
I
120
I
I
I
240
rnin of post-TS160-treatrnent Fig. 3.
Figs. 1, 2 and 3. The course of DNA synthesis (Fig. 1), RNA synthesis (Fig. 2) and protein synthesis (Fig. 3) in control V79 cells and in the cells treated with TSI60 for 60 rain. O, 0.5/~g/ml; t,, 1/~g/ml; A, 3 #g/ml; I, 5 #g/ml; D, 10 #g/ml; o, control. mutagenic effect is not dependent on the manner of treatment, but is in close relationship with the level of survival. The frequency of 6-TG r mutations increased in the span of survival from 90 to 10%; however, when the surviving fraction fell below 10%, the cytotoxic effects of TS16o covered the mutagenic effects and the frequency of 6-TG r mutations gradually decreased. When the survival was 1% or less we could not find any induced 6-TG r mutations in the population of surviving cells. The rather low mutagenic effects of TS160 found in V79 cells after a single treatment (Fig. 6) led us to test the effect of repeated treatment with this substance. We found that 4 treatments with TS~60, at 8-day intervals, increased the sensitivity of V79 cells to this substance only partly, while the mutagenic effects of the 4 treatments had a roughly additive character (Table 1). It is interesting to compare this result with those obtained from experiments with chronic treatment of rats with N, N-dimethylnitrosamine ( D M N ) (Montesano et al., 1980), where the livers of treated animals augmented their ability to repair O6-meth ylguanine (i.e., D N A damage), which is considered, in respect of mutagenic effects, to be the most significant. Thus, a decreased rather than an increased mutagenic effect following repeated doses of the alkylating agent could be expected. However, as discussed below, production of O6-alkylguanine is not the only D N A damage caused by TSI6o. Similarly, the highly specific properties of liver cells can hardly be generalized.
106
1
days after
treatment
Fig. 4. Growth activity of V79 cells after 60 min incubation with various concentrations of TSI60. O, 0.5 /.tg/ml; A, 1 /~g/ml; &, 3 /~g/ml; I , 5 /~g/ml; D, 10 btg/ml; e, control.
100 -
~,,,,,
0"'-
-o..p 0
o
, ,,
o'" "0"'-% °
o
0
\
10. It,. O
\ \ \, tn t
.-e
-0
\
'~x t \ I
\ \ t
I
I I
0
Y!
6s
I 4ag TS160/m[
Fig. 5. Colony-forming ability of V79 cells after 60 rain incubation with various concentrations of TS:6 o in complete medium (O), and after 30 min of incubation in a medium without serum (zx) and in PBS buffer
(D).
437
10
o =8
m
~7 '%
bi
~
?
? i
'
6
8
4
z~
3 I
I1~
100
|
|
I
I
I
|11
10
i1!
1
i
o/. OF
|
i
iii
1 SURVIVORS
i
i~
iimiii
0.1
Fig. 6. Frequency of induced 6-TG r mutations in V79 cells treated with various concentrations of TSt60 in complete medium (60 min, ©), in medium without serum (30 min, zx) and in PBS buffer (30 min, n). The individual points represent mean values obtained from 3 different Expts. For determination of 1 point 2.5 × 10 6 cells from 5 petri dishes were tested.
'
The resistance of several spontaneous 6-TG r cell lines t o TS]60 was observed in further experiments to eliminate the possibility that TSI60 had not induced, but had selected, 6-TG r mutations. Our results showed that spontaneous 6-TG r cells (isolated from V79 cells) can be characterized by the same or lower resistance to TS~60 than the V79 cells. The selection of spontaneous 6-TG r mutations by TSI60 is therefore not probable. How can we explain the mutagenicity of TS~60? Alkylating agents usually attach to D N A in the N-7 position of guanine. These modifications of guanine had therefore been considered to be the source of mutations. It was established, however (Lawley, 1974), that, with respect to mutagenicity, the alkylation of guanine in the 0 - 6 position was more important because it changed the pairing capacity of guanine. F r o m this aspect, alkylation in further positions of D N A bases (0-4 thymine; N-3 guanine; N-1 and N-7 adenine; N-3 cytosine and N-3 and N-4 thymine) must be important (Auerbach, 1976). It is necessary to emphasize that the induction of mutations is only a rare event, so that only few cases of alkylation result in mutation. The secondary effects of alkylation have to be considered as the other alternative for the origin of mutations in alkylated DNA. As mentioned above, N-glycosidic bonds are unstable in guanines alkylated in N-3 and N-7 positions. This situation m a y result in creation of apurinic sites or further in single-strand breaks of DNA. After alkylation of D N A by bifunctional or polyfunctional agents ( H N 2, T S l 6 0 ) ,
0 1 1.5 1.75 2 2.25 2.5 2.75 3
S e c o n d treatment
O 1 1.5 1.75 2 2.25 2.5 2.75 3
First treatment
TSI6o (/~g/ml)
81.8 63.2 68.5 40.4 44 40.1 30.6 29.9 20.5
96 62.7 55.8 46.3 42.5 25.8 28.9 25.2 25
PE (AT)
74.6 59.8 82.3 73.4 91.1 78.8 95.9 68.7 74
0.164+0.1313 1.070 -+ 1.2641 1.798 + 0.6262 1.089 + 0.3337 1.229+0.1075 1.218 + 0.6663 0.959 + 0.5374 1.647 -+0.9497 8.702 + 0.8614
1 6.52 10.69 6.64 7.49 7.42 5.84 10.05 53.06
R
72.7 83.1 86.9 61.0 76.9 99.9 71.0 68.8 105.6
PE (%)
M -+ o
89.6 92.1 80.8 89.5 73.2 89.3 70.2 85.7 76.5
PE (%)
1 4.68 3.29 13.09 2.02 3.69 2.92 9.28 21.97 11 days
0.154±0.1244 0.722±0.5476 0.508 -+0.3218 2.017 -+0.5814 0.312-+0.2846 0.569 -+0.1163 0.450-+0.3820 1.430-+0.3103 3.389 -+0.8228
8 days
77.6 99.6 78.6 81.3 102.2 84.3 87.6 69.9 86.3
PE (%)
R
PE (%)
M± o
9 days
7 days
Expression time
OCCURRENCE OF 6-TG r MUTATIONS IN V79 CELLS AFTER 1, 2 A N D 4 TREATMENTS WITH TSI6o
TABLE 1
1
12.29 6.97 26.03
3.380 _+0.9257 1.910 +0.6527 7.154 +0.5213
1
7.96 10.56 9.29 2.7 1.5 3.2 7.07 10.28
2.27 4.74 11.19 2.08
-+0.2255 +0.9613 -+0.9981 +0.7915 -+0.5080 -+0.2194 -+0.4029 ± 0.5622 -+0.6186
_+0.2460 + 0.2454 _+0.4153 _+ 1.2494 _+0.3176
0.275 0.625 1.304 3.079 0.572
M_.+o
0.178 1.52 1.881 1.653 0.491 0.268 0.569 1.259 1.830
M-+o
O~
17.2 15.7 13.5 lO
2.25 2.5 2.75 3
. 74.1 89.1 92.6
90.8 79.8 61.6 73.9 80.3 .
. 1.942 ± 0.6378 6.690 ± 0.6912 20.605±2.2782
0.149 + 0.1837 1.203 _+0.4010 3.051 + 0 . 7 8 4 6 1.510 +_0.4220 2.760+0.2672 . 13.03 44.91 138.25
1 8.07 20.47 10.13 18.52 . 93 51.4 64.2
83.9 58 82.9 91.5 71.7 24.534 ± 1.3618
0.2010+0.1786 1.655 _+0.5500 3.570 + 1.0393 3.730 _____1.0509
M± o
x i is the sum of n u m b e r s of 6 - T G r m u t a n t s per 100000 cells in i n d i v i d u a l samples, p is the m e a n of these values, n is their number. R , ratio of i n d u c e d to s p o n t a n e o u s m u t a t i o n s .
P E (AT), p l a t i n g efficiency i m m e d i a t e l y after treatment. M ± o, average n u m b e r s of 6 - T G r m u t a n t s per 100000 cells + s t a n d a r d d e v i a t i o n o. The s t a n d a r d d e v i a t i o n o was c a l c u l a t e d by the e q u a t i o n
86.6 71.8 42.2 41.7 25
Fourth treatment
0 1 1.5 1.75 2
P E (%)
R
PE (%)
M± o
8 days
6 days
122.03
18.59
1 8.23 17.76
".O
440 often cross-links between the DNA strands (mostly between the guanines in N-7 positions) may be formed. These can be excised spontaneously or enzymatically, l e a v i n g d o u b l e - s t r a n d b r e a k s i n t h e D N A . I t is p o s s i b l e t h a t t h e s e c o n d a r y m u t a g e n i c e f f e c t s o f D N A a l k y l a t i o n r e s i d e i n i r r e g u l a r f i l l i n g u p o f t h e a p u r i n i c sites i n DNA
or in erroneous repair of single- or double-strand breaks in DNA.
Acknowledgements We are greatly indebted solicitous technical help.
t o M r s . A. M o r / t v k o v ~ a n d M r s . H . P l ~ t e n i k o v ~ f o r
References Anderson, D., and M. Fox (1974) The induction of thymine and IUdR resistant variants in P388 mouse lymphoma cells by X-rays, UV and mono- and bi-functional alkylating agents, Mutation Res., 25, 107-122. Auerbach, C. (1976) The chemical mutagens: alkylating agents, in: Chapman and Hall (Eds.), Mutation Research; Problems, Results and Perspectives, A Halsted Press Book, John Wiley, New York, Ch. 16. Griffin, A.C., E.L. Brandt and E.L. Tatum (1951) Induction of tumors with nitrogen mustards, Cancer Res., 11,253-260. Heston, W.E. (1953) Occurrence of tumors in mice injected subcutaneously with sulphur mustard and nitrogen mustard, J. Natl. Cancer Inst., 14, 131-148. Lawley, P.D. (1974) Some chemical aspects of dose-response relationships in alkylation mutagenesis, Mutation Res., 23, 283-295. Lawley, P.D., and P. Brookes (1963) Further studies on the alkylation of nucleic acids and the constituent nucleotides, Biochem. J., 89, 127-138. O'Connor, P.J. (1981) Studies on mechanism of action; Interaction of chemical carcinogens with macromolecules, J. Cancer Res. Clin. Oncol., 99, 167-186. Montesano, R., H. Br/:sil, G. Planche-Martel, G.P. Margison and A.E. Pegg (1980) Effect of chronic treatment of rats with dimethylnitrosamine on the removal of O6-methylguanine from DNA, Cancer Res., 40, 452-458. Shimkin, M.B., J.H. Weisburger, E.K. Weisburger, N. Gubareff and V. Suntzeff (1966) Bioassay of 29 alkylating chemicals by the pulmonary-tumor response in strain A mice, J. Natl. Cancer Inst., 36, 915-935. Slamefiovb., D., and A. Gabelovh (1980) The effects of sodium azide on mammalian cells cultivated in vitro, Mutation Res., 71,253-261. Slamefiovb,, D., A. Gabelovb. and M. Kothajovb. (1980) Notes to application of the DNA synthesis inhibition test in the screening of DNA-damaging factors, Studia Biophys., 78, 3, 165-175. Suter, W., J. Brennand, S. McMillan and M. Fox (1980) Relative mutagenicity of antineoplastic drugs and other alkylating agents in V79 Chinese hamster cells, independence of cytotoxic and mutagenic response, Mutation Res., 73, 171-181. S~'kora, I., V. Vortel, O. Marhan and A. Dynterovh (1981) Carcinogenicity of trichlormethine hydrochloride (ZSl60 Spofa) and morphological damage after its intraamniotic injection, Neoplasma. 28, 5, 565-574.