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The cytotoxic and mutagenic effects of 4CMB, BC and 4HMB in V79 Chinese hamster cells
Mutation Research, 100 (1982) 239-244
239
Elsevier Biomedical Press
THE CYTOTOXIC AND MUTAGENIC EFFECTS OF 4CMB, BC AND 4HMB IN V79 CHINESE HAMSTER CELLS
FARIDEH MIRZAYANS, P.J. DAVIES and JAMES M. PARRY Department of Genetics, University College of Swansea, Singleton Park, Swansea SA2 8PP (Great Britain)
(Received 10 April 1981) (Accepted 20 May 1981)
SUMMARY 4CMB and BC were shown to be capable of inducing mutation in V79 Chinese hamster ceils whereas no such activity was detected after treatment with 4HMB.
BC, 4CMB and 4 H M B were tested for their cytotoxicity and their ability to induce mutation at the H G P R T locus and for resistance to the steroid compound ouabain in a V79 (H) (supplied by John Thacker - Harwell) Chinese hamster lung fibroblast cell line. The techniques used have been described in detail by Arlett et al. (1972, 1975) except that Dulbecco's modified Eagles medium (DMEM) with 10070 foetal calf serum was used. All these chemicals were dissolved in dimethyl sulphoxide (DMSO) and the final concentration of DMSO in media at all times was 1070, which was routinely added to DMSO control plates. Exponentially growing V79 (H) cultures were exposed to the test chemicals at various concentrations and the media containing chemicals were then removed after a 1-h treatment period. Cultures were washed with PBS and plated out at 105 cells per 9-cm petri dishes for mutant isolation. The selective agents 8-azaguanine (8AZG), 6-thioguanine (6TG) and ouabain (Oua) were added to give final concentrations of 30/~g/ml, 5/~g/ml and 0.1 m M respectively, at different intervals to give expression times of 24, 48, 56, 68 and 75 h after chemical treatment. Treated and untreated cultures were also plated out at the lower cell density of 102 and 103 cells per 5-cm petri dishes to estimate cell viability in the presence and absence of the test chemicals. Cultures were then incubated at 37 ° in a CO2
Abbreviation: PBS, phosphate-buffered saline.
0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
240
atmosphere for 7 days and 14 days to measure cell survival and mutation induction respectively. Cultures were subsequently stained with methylene blue solution and scored. RESULTS
Fig. 1 shows the cell toxicity after a 1-h exposure to 4CMB, BC and 4HMB with various concentrations of 4CMB, BC and 4HMB. (10%0 survival was obtained after treatment with 170 #M 4CMB and 280 #M BC. 4HMB was not toxic over the 1-h treatment period.) 4HMB at concentrations greater than 1000/zM precipitated in the media. A 4-h treatment with 1000 #M 4HMB resulted in 50% cell killing (data now shown).
100-
0
\ .>
10 d~
o
..I
0
,oo
2oo
3oo
Concentration ~M) Fig. 1. Survival of V79 cells after 1-h treatment with various concentrations of 4CMB ( • ) , BC (O) and 4HMB (A).
241
0,p03
. 04 oo
8 o
LL
1O0
200
Concentration ( pM )
Fig. 2. Dose-response curves showing the frequency of induced 8AZG R mutants/105 survivors after l-h treatment with 4CMB ( e ) , BC (O) and 4HMB (A).
Fig. 2 shows the frequency of induced 8AZG-resistant mutants per 105 survivors after a 1-h treatment with 4CMB, BC and 4HMB. The data demonstrate that both 4CMB and BC were mutagenic in V79 (H) cells whereas 4HMB did not induce any mutation above the spontaneous levels. On the basis of the frequency of 8AZGresistant mutants against concentration little difference could be seen between the 2 genetically active compounds. However, when these data were plotted as induced mutants against toxicity as in Fig. 3, then it can be seen that at equivalent toxicity BC was the more active compound. Cell viability, mutagenicity plate counts and mutation frequencies induced by 4CMB, BC and 4HMB at HGPRT locus
u3 0
O. ,1¢
"E ¢0
OC)O0
o
j
J
J"
A IJ.
100
4i~ Log. % Survival
Fig. 3. Frequency of induced 8AZG R mutants/105 survivors in V79 cells after treatment with equitoxic concentrations of 4CMB (O), BC (O) and 4HMB (2~).
242 TABLE I P L A T I N G EFFICIENCY, M E A N N U M B E R OF 8AZG-RESISTANT M U T A N T S PER P L A T E AT O P T I M U M EXPRESSION TIMES, 8AZG-RESISTANT M U T A N T S PER 105 SURVIVORS A N D F R E Q U E N C Y OF I N D U C E D 8AZG-RESISTANT M U T A N T S P E R 105 SURVIVORS IN UNT R E A T E D A N D T R E A T E D V79 (H) CELLS W I T H V A R I O U S C O N C E N T R A T I O N S OF 4CMB A N D BC FOR 1 h A N D W I T H 1000 #M 4 H M B FOR 2, 3 A N D 4 h Each point is the average of at least 5 plates. Conc. (~M)
Mean number of viable cells/ 102 plated
Mean n u m b e r of 8AZGRmutants per plate treated
4CMB
BC
4HMB 2h 3h 4h
0 50 75 100 120 200
109 117.2 80;7 54.5 24.8 4.3
22.5 16.7 29.3 22.3 17.5
80 150 200
99.6 65.3 43
54.3 61.8 184.3
1000 1000 1000
84.3 92.5 69.2
59 49.2 50
8AZGRmutants/ 105 survivors
Induced mutation frequency/105 survivors
treated
untreated
20 5 20.7 0 27.5
19.1 20.7 53.9 90.4 404.1
18.9 5.3 20.5 0 26
0.2 15.3 33.4 90.4 378.1
33 43 53
54.4 99.6 428.6
27.9 41.9 55.9
26.6 57.7 372.6
69.9 53.1 72.2
50.7 22.7 38.3
19.2 30.4 33.9
untreated
45.6 27.3 46
U3 0,¢--
i
1== 100 Concentration ( pM ) Fig. 4. Dose-response curves showing the frequency o f induced O u a R m u t a n t s / l O 5 survivors after 1-h treatment with 4CMB ( o ) and BC (O).
243
i
%
i 16o Concentration ( pM ) Fig. 5. Dose-response curves showing the frequency of induced 6TG R mutants/105 survivors after t-h treatment with 4CMB ( e ) and BC (O).
(hypoxanthine guanine phosphoribosyl-transferase) are shown in Table 1. The mutation induction of 4CMB and BC was also determined for induced resistance to ouabain and 6-thioguanine. The results are shown in Figs. 4 and 5, which demonstrate that both compounds were capable of inducing mutation at both loci. However, since the mean number of colonies per plate in treated cultures was almost the same as control plates (not more than 2 colonies/plate at any expression time), from these results no statistically significant conclusions can be drawn. CONCLUSIONS
Both BC and 4CMB were shown to be capable of inducing mutation at the HGPRT locus for V79 cells with BC showing the greater genetic activity at equivalent levels of toxicity, whereas no such activity was detectable with 4HMB. BC and 4CMB also gave increased levels of mutation at the 6TG- and Oua-sensitive loci but the small number of colonies observed in the experiments prevented an adequate interpretation of the data. ACKNOWLEDGEMENTS
The work was in part supported by funds made available by the EEC Environmental Programme. We would like to thank Razmik Mirzayans for his advice and encouragement.
244 REFERENCES Arlett, C.F., and S.A. Harcourt (1972) Expression time and spontaneous mutability in the estimation of induced mutation frequency following treatment of Chinese hamster cells by ultraviolet light, Mutation Res., 16, 301-306. Arlett, C.F., D. Turnbull, S.A. Harcourt, A.R. Lehmann and C.M. ColeUa (1975) A comparison of the 8-azaguanine- and ouabain-resistance systems for the selection of induced mutant Chinese hamster cells, Mutation Res., 33,261-278.
239
Elsevier Biomedical Press
THE CYTOTOXIC AND MUTAGENIC EFFECTS OF 4CMB, BC AND 4HMB IN V79 CHINESE HAMSTER CELLS
FARIDEH MIRZAYANS, P.J. DAVIES and JAMES M. PARRY Department of Genetics, University College of Swansea, Singleton Park, Swansea SA2 8PP (Great Britain)
(Received 10 April 1981) (Accepted 20 May 1981)
SUMMARY 4CMB and BC were shown to be capable of inducing mutation in V79 Chinese hamster ceils whereas no such activity was detected after treatment with 4HMB.
BC, 4CMB and 4 H M B were tested for their cytotoxicity and their ability to induce mutation at the H G P R T locus and for resistance to the steroid compound ouabain in a V79 (H) (supplied by John Thacker - Harwell) Chinese hamster lung fibroblast cell line. The techniques used have been described in detail by Arlett et al. (1972, 1975) except that Dulbecco's modified Eagles medium (DMEM) with 10070 foetal calf serum was used. All these chemicals were dissolved in dimethyl sulphoxide (DMSO) and the final concentration of DMSO in media at all times was 1070, which was routinely added to DMSO control plates. Exponentially growing V79 (H) cultures were exposed to the test chemicals at various concentrations and the media containing chemicals were then removed after a 1-h treatment period. Cultures were washed with PBS and plated out at 105 cells per 9-cm petri dishes for mutant isolation. The selective agents 8-azaguanine (8AZG), 6-thioguanine (6TG) and ouabain (Oua) were added to give final concentrations of 30/~g/ml, 5/~g/ml and 0.1 m M respectively, at different intervals to give expression times of 24, 48, 56, 68 and 75 h after chemical treatment. Treated and untreated cultures were also plated out at the lower cell density of 102 and 103 cells per 5-cm petri dishes to estimate cell viability in the presence and absence of the test chemicals. Cultures were then incubated at 37 ° in a CO2
Abbreviation: PBS, phosphate-buffered saline.
0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
240
atmosphere for 7 days and 14 days to measure cell survival and mutation induction respectively. Cultures were subsequently stained with methylene blue solution and scored. RESULTS
Fig. 1 shows the cell toxicity after a 1-h exposure to 4CMB, BC and 4HMB with various concentrations of 4CMB, BC and 4HMB. (10%0 survival was obtained after treatment with 170 #M 4CMB and 280 #M BC. 4HMB was not toxic over the 1-h treatment period.) 4HMB at concentrations greater than 1000/zM precipitated in the media. A 4-h treatment with 1000 #M 4HMB resulted in 50% cell killing (data now shown).
100-
0
\ .>
10 d~
o
..I
0
,oo
2oo
3oo
Concentration ~M) Fig. 1. Survival of V79 cells after 1-h treatment with various concentrations of 4CMB ( • ) , BC (O) and 4HMB (A).
241
0,p03
. 04 oo
8 o
LL
1O0
200
Concentration ( pM )
Fig. 2. Dose-response curves showing the frequency of induced 8AZG R mutants/105 survivors after l-h treatment with 4CMB ( e ) , BC (O) and 4HMB (A).
Fig. 2 shows the frequency of induced 8AZG-resistant mutants per 105 survivors after a 1-h treatment with 4CMB, BC and 4HMB. The data demonstrate that both 4CMB and BC were mutagenic in V79 (H) cells whereas 4HMB did not induce any mutation above the spontaneous levels. On the basis of the frequency of 8AZGresistant mutants against concentration little difference could be seen between the 2 genetically active compounds. However, when these data were plotted as induced mutants against toxicity as in Fig. 3, then it can be seen that at equivalent toxicity BC was the more active compound. Cell viability, mutagenicity plate counts and mutation frequencies induced by 4CMB, BC and 4HMB at HGPRT locus
u3 0
O. ,1¢
"E ¢0
OC)O0
o
j
J
J"
A IJ.
100
4i~ Log. % Survival
Fig. 3. Frequency of induced 8AZG R mutants/105 survivors in V79 cells after treatment with equitoxic concentrations of 4CMB (O), BC (O) and 4HMB (2~).
242 TABLE I P L A T I N G EFFICIENCY, M E A N N U M B E R OF 8AZG-RESISTANT M U T A N T S PER P L A T E AT O P T I M U M EXPRESSION TIMES, 8AZG-RESISTANT M U T A N T S PER 105 SURVIVORS A N D F R E Q U E N C Y OF I N D U C E D 8AZG-RESISTANT M U T A N T S P E R 105 SURVIVORS IN UNT R E A T E D A N D T R E A T E D V79 (H) CELLS W I T H V A R I O U S C O N C E N T R A T I O N S OF 4CMB A N D BC FOR 1 h A N D W I T H 1000 #M 4 H M B FOR 2, 3 A N D 4 h Each point is the average of at least 5 plates. Conc. (~M)
Mean number of viable cells/ 102 plated
Mean n u m b e r of 8AZGRmutants per plate treated
4CMB
BC
4HMB 2h 3h 4h
0 50 75 100 120 200
109 117.2 80;7 54.5 24.8 4.3
22.5 16.7 29.3 22.3 17.5
80 150 200
99.6 65.3 43
54.3 61.8 184.3
1000 1000 1000
84.3 92.5 69.2
59 49.2 50
8AZGRmutants/ 105 survivors
Induced mutation frequency/105 survivors
treated
untreated
20 5 20.7 0 27.5
19.1 20.7 53.9 90.4 404.1
18.9 5.3 20.5 0 26
0.2 15.3 33.4 90.4 378.1
33 43 53
54.4 99.6 428.6
27.9 41.9 55.9
26.6 57.7 372.6
69.9 53.1 72.2
50.7 22.7 38.3
19.2 30.4 33.9
untreated
45.6 27.3 46
U3 0,¢--
i
1== 100 Concentration ( pM ) Fig. 4. Dose-response curves showing the frequency o f induced O u a R m u t a n t s / l O 5 survivors after 1-h treatment with 4CMB ( o ) and BC (O).
243
i
%
i 16o Concentration ( pM ) Fig. 5. Dose-response curves showing the frequency of induced 6TG R mutants/105 survivors after t-h treatment with 4CMB ( e ) and BC (O).
(hypoxanthine guanine phosphoribosyl-transferase) are shown in Table 1. The mutation induction of 4CMB and BC was also determined for induced resistance to ouabain and 6-thioguanine. The results are shown in Figs. 4 and 5, which demonstrate that both compounds were capable of inducing mutation at both loci. However, since the mean number of colonies per plate in treated cultures was almost the same as control plates (not more than 2 colonies/plate at any expression time), from these results no statistically significant conclusions can be drawn. CONCLUSIONS
Both BC and 4CMB were shown to be capable of inducing mutation at the HGPRT locus for V79 cells with BC showing the greater genetic activity at equivalent levels of toxicity, whereas no such activity was detectable with 4HMB. BC and 4CMB also gave increased levels of mutation at the 6TG- and Oua-sensitive loci but the small number of colonies observed in the experiments prevented an adequate interpretation of the data. ACKNOWLEDGEMENTS
The work was in part supported by funds made available by the EEC Environmental Programme. We would like to thank Razmik Mirzayans for his advice and encouragement.
244 REFERENCES Arlett, C.F., and S.A. Harcourt (1972) Expression time and spontaneous mutability in the estimation of induced mutation frequency following treatment of Chinese hamster cells by ultraviolet light, Mutation Res., 16, 301-306. Arlett, C.F., D. Turnbull, S.A. Harcourt, A.R. Lehmann and C.M. ColeUa (1975) A comparison of the 8-azaguanine- and ouabain-resistance systems for the selection of induced mutant Chinese hamster cells, Mutation Res., 33,261-278.