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Mutagenic activity of 3 diazoacetylglycine derivatives on V79 Chinese hamster cells
375
Mutation Research, 78 (1980) 375--379 © Elsevier/North-Holland Biomedical Press
MUTAGENIC ACTIVITY OF 3 DIAZOACETYLGLYCINE DERIVATIVES ON V79 CHINESE HAMSTER CELLS
B. PANI, N. BABUDRI, F. BARTOLI-KLUGMANN a, S. VENTURINI and I. di FANT a Institute of Microbiology, University of Trieste, Trieste (Italy), and a Institute of Pharmacology, University of Trieste, Trieste (Italy)
(Received 29 November 1979) (Revision received 26 February 1980) (Accepted 14 March 1980) Summary 3 diazoacetylglycine derivatives, diazoacetylglycine amide {DGA), diazoacetylglycine ethyl ester (DGE) and diazoacetylglycine hydrazide (DGH), known as antitumour agents, and shown to be mutagenic in bacteria, were studied for their mutagenic activity in the H G P R T system of V79 Chinese hamster cells in culture ( V 7 9 / H G P R T system). All 3 drugs were highly effective in inducing 6-thioguanine-resistant mutants at concentrations that were not significantly cytotoxic.
Diazoacetylglycine amide (DGA), diazoacetylglycine ethyl ester (DGE) and diazoacetylglycine hydrazide (DGH), known as antitumour agents {Baldini and Brambilla, 1966), have shown a powerful mutagenic activity for strain T A 1 5 3 5 of S a l m o n e l l a t y p h i m u r i u m , a strain reverted b y base-pair substitution inducing agents, b u t no mutagenic activity for 3 other strains (TA1536, TA1537, TA1538), which contain frameshift mutations (Monti-Bragadin et al., 1974). Furthermore, the mutagenic activity of these c o m p o u n d s was more marked in strains of S a l m o n e l l a t y p h i m u r i u m carrying a defect in this excision-repair system (Banff et al., 1974). These observations are compatible with the idea that diazoacetylglycine derivatives display this activity through alkylation of bacterial DNA. Bacteria differ from mammalian cells in the organization of genetic material, cell permeability, metabolism and presumably in the complexity of DNA-repair processes; thus, mutagenic response to chemicals is n o t necessarily expected to be identical in bacteria and eucaryotic cells. Therefore, to investigate the genotoxic activity of diazoacetylglycine derivatives, we tested DGA, DGE and DGH for their mutagenic activity on V79 Chinese hamster cells in culture, using the hypoxanthine--guanine-phosphoribosyl transferase deficiency system (HGPRT-).
376 CO--CH=N2 I
NH I CH2
~ DGE O - C H 2 - C H 3
;
R ~ DGA~NH2
L
I
co-R
DGI~NH_NH2
Fig. 1. S t r v c t u r e o f t h e 3 d i a z o a c e t y l g l y c i n e d e r i v a t i v e s .
DGE
DGA ~u z :::) O
DGI
2000
1500
N M.
~"
1000
-~ ~
sap
-~ Q ~,
~
3
5
~
3
7 EXPRESSION
5 TIME
7
3
S
~
IDAYS)
Fig. 2. E x p r e s s i o n t i m e of T G - r e s i s t a n t p h e n o t y p e . M u t a n t s p e r m i l l i o n viable cells axe p l o t t e d a g a i n s t e x p r e s s i o n t i m e in d a y s a f t e r t h e t r e a t m e n t w i t h 5 m M ( e ) a n d 10 m M (o) D G A , D G E a n d D G H . P o i n t s r e p r e s e n t the m e a n v a l u e s f r o m 2 i n d e p e n d e n t E x p t s . V e r t i c a l b a r s i n d i c a t e s t a n d a r d e r r o r s of the m e a n s (controls have been subtracted).
-: b) ~ ~
2000.
~,~ z ~ ~0o ~. ~~
~
~.
~.
A
I
25 8 DOSE/IO ~ ~
-
a)
I
10
Fig. 3. M u t a t i o n f r e q u e n c y (a) ~ d s u ~ i v ~ (b) a f t e r t r e a t m e n t w i t h d i ~ o a c e t y l ~ y c i n e d e ~ v a t i v e s D G A , D G E a n d D G H , as a ~ u n c t i o n o f t h e c o n c e n t r a t i o n . P o ~ t s ~ e the a v e r a g e f~om 4 i n d e p e n d e n t R x p t s . , a n d v e r t i c ~ b a r s r e p r e ~ n t the s t ~ d ~ d e r r o r s of the m e a n s ( c o n t r o l s h a v e b e e n s u b t r a c t e d ) , e , D G A ; a, D G E : ~, D G H .
377 Material and m e t h o d s Cell culture V79 Chinese hamster cells, originally obtained from Prof. Abbondandolo (Laboratorio di Mutagenesi e Differenziamento, CNR, Pisa) were grown in Dulbecco's minimal essential medium (DMEM), supplemented with 10% foetal calf serum (Microbiological Associates), penicillin at 100 IU/ml and streptomycin {Squibb) at 100 pg/ml, and were maintained in a humidified 5% CO: atmosphere at 37°C. Trypsin {1 : 250 Difco) at 0.25% in Dulbecco's phosphate-buffered saline (D-PBS) was routinely used for subculture. A clone of V79 cells, designated V79/D4, selected in our laboratory for its high plating efficiency and its low spontaneous mutation rate, was used throughout this work. Chemicals DGA, DGE and DGH (Fig. 1) were synthesized and kindly supplied by Prof. L. Baldini, Istituto di Farmacologia, Universit~ di Trieste. The 2-aminomercaptopurine (6-thioguanine, TG) was purchased from Sigma. Cell treatment V 7 9 / D 4 cells (2.5--3 × 106) were plated in 50-mm diameter dishes and incubated for 24 h at 37°C before the addition of the treatment solutions. The culture medium was replaced with fresh medium 1 h before the treatment. Cells were incubated for 1 h in 2.5, 5 and 10 mM solutions of each comp o u n d in medium without serum. Untreated controls were included in each experiment. At the end of the treatment, all the cultures were washed 4 times with medium without FBS. Cell survival and mutagenesis assay After mutagenic treatment, cells were detached from each dish with trypsin and the density of the resulting single~ell suspension was estimated in a haemocytometer. 5 × l 0 s cells were seeded in non-selective medium to propagate the culture from each expression time to the next (Abbondandolo et al., 1976). For the determination of the induced toxicity, 2 × 10: cells/50-mm dish, and for mutagenesis 2 × 10 s cells/100-mm dish, were plated. 1 h after the plating, 6-TG was added to the mutation plates at the final concentration of 4 pg/ml. To achieve maximal recovery of mutants, a complete expression time curve was constructed for 5 and 10 mM DGA, DGE and DGH. In practice, 5 replicates were treated with 6-TG at each of the 4 expressiontime points ranging from 0 to 7 days after treatment. Both survival and mutation plates were stained with 1% methylene blue 7 days after the plating, and colonies of more than a b o u t 50 cells were scored macroscopically. Results
The treatment of V 7 9 / D 4 cells with 2.5, 5 and 10 mM DGA, DGE or D G H induced a significantly higher number of 6-TG-resistant mutants than untreated controls, which never exceeded 30 mutants per 106 viable ceils.
378 Fig. 2 shows the frequency of 6-TG-resistant clones when the selective agent was added at various times after treatment with diazoacetylglycine derivatives. It is evident that a 5-day period was necessary for the expression of the maximal number of mutants. This is presumably the time necessary for the decay of pre-existing H G P R T enzyme and m R N A , which would occur through normal turnover mechanisms as well as dilution by cell division (Van Zeeland and Simons, 1976). In Fig. 3 the mutation frequency at 5
379
DGA >~ DGE > DGH. Furthermore, in the HGPRT system, the DGH is much less effective than the other derivatives, perhaps indicating a lower reactivity of the molecule carrying the hydrazide group. Acknowledgements We are grateful to Prof. Carlo Monti-Bragadin for useful discussions and to Prof. A. Abbondandolo (Laboratorio di Mutagenesi e Differenziamento, CNR, Pisa) for V79 cells. The technical assistance of Mrs. Presti and Mr. Gamboz is acknowledged. References Abbondandolo0 A., S. Bonatti, C. Colella, G. Corti, F. Matteucci, A. Mazzacaro and G. Rainaldi (1976) A comparative study of different e x p e r i m e n t a l protocol s for mutagenesis assays with 8-azaguanine resistance system in Chinese hamster cultured ceils, Mutation Res., 37, 293--306. Baldini, L., and G. Brambilla (1966) Antineoplastic activity of diazoacetylglycine derivatives, Cancer Res., 26, 1754--1758. Banfi, E., M. Tamaro, B. Pani and C. Monti-Bragadin (1974) Mechanism of the mutagenic activity of diazoacetylglycine derivatives, Boil. Ist. Sieroter. Milanese0 53, 632--635. Brambilla, G., S. Parodi, M. Cavanna and L. Baldini (1970) The immunodepressive activity of N-diazoacetylglyctne amide in some transplantation systems, Transplantation, 10, 100--105. Brambilla, G., M. Cavanna, S. Parodi and L. Baldini (1972) I n d u c t i o n of t umors in n e w b o r n and a dul t Swiss mice by N-diazoacetylglycine amide, Eur. J. Cancer, 8, 127--129. Gixaldi, T., and L. Baldini (1973) Mechanism of inhibition of DNA synthesis in Ehrlich ascites t u m o u r ceils by diazoacetylglycine amide, Biochem. Pharmacol., 22, 1793--1799. Monti-Bragadin, C., M. Tama~o and E. Banff (1974) Mutagenic activity of diazoacetylglycine derivatives, An timicrob. Agents Chemother., 6, 655--656. Parodi, S., C. Bolognesi, M. Cavanna, R.L. Pollack, L. Santi and G. Brambilla (1977a) Damage and repair of DNA in cultured m a m m a l i a n ceils with N-diazoacetylglycine amide, Cancer Res., 37, 4460---4466. Parodi, S., M. Picca, C. Bolognesi, M. Cavanna, P. Carlo, R. Finollo and G. Brambilla (1977b) A l k y l a t i o n of 7-(4-nitrobenzyl)pyridine by four N-diazoacetyl-derivatives of amino acids, Lack of fine correlation with the unscheduled DNA synthesis induced in mouse ceils "in vi t ro" , Pharmacol. Res. Commun.. 9,621-631. Van Zeeland, A.A., and J.W.I.M. Simons (1976) Linear dose--response relationships after prolonged expression times in V79 Chinese hamster ceils, Mutation Res., 35, 129--138.
Mutation Research, 78 (1980) 375--379 © Elsevier/North-Holland Biomedical Press
MUTAGENIC ACTIVITY OF 3 DIAZOACETYLGLYCINE DERIVATIVES ON V79 CHINESE HAMSTER CELLS
B. PANI, N. BABUDRI, F. BARTOLI-KLUGMANN a, S. VENTURINI and I. di FANT a Institute of Microbiology, University of Trieste, Trieste (Italy), and a Institute of Pharmacology, University of Trieste, Trieste (Italy)
(Received 29 November 1979) (Revision received 26 February 1980) (Accepted 14 March 1980) Summary 3 diazoacetylglycine derivatives, diazoacetylglycine amide {DGA), diazoacetylglycine ethyl ester (DGE) and diazoacetylglycine hydrazide (DGH), known as antitumour agents, and shown to be mutagenic in bacteria, were studied for their mutagenic activity in the H G P R T system of V79 Chinese hamster cells in culture ( V 7 9 / H G P R T system). All 3 drugs were highly effective in inducing 6-thioguanine-resistant mutants at concentrations that were not significantly cytotoxic.
Diazoacetylglycine amide (DGA), diazoacetylglycine ethyl ester (DGE) and diazoacetylglycine hydrazide (DGH), known as antitumour agents {Baldini and Brambilla, 1966), have shown a powerful mutagenic activity for strain T A 1 5 3 5 of S a l m o n e l l a t y p h i m u r i u m , a strain reverted b y base-pair substitution inducing agents, b u t no mutagenic activity for 3 other strains (TA1536, TA1537, TA1538), which contain frameshift mutations (Monti-Bragadin et al., 1974). Furthermore, the mutagenic activity of these c o m p o u n d s was more marked in strains of S a l m o n e l l a t y p h i m u r i u m carrying a defect in this excision-repair system (Banff et al., 1974). These observations are compatible with the idea that diazoacetylglycine derivatives display this activity through alkylation of bacterial DNA. Bacteria differ from mammalian cells in the organization of genetic material, cell permeability, metabolism and presumably in the complexity of DNA-repair processes; thus, mutagenic response to chemicals is n o t necessarily expected to be identical in bacteria and eucaryotic cells. Therefore, to investigate the genotoxic activity of diazoacetylglycine derivatives, we tested DGA, DGE and DGH for their mutagenic activity on V79 Chinese hamster cells in culture, using the hypoxanthine--guanine-phosphoribosyl transferase deficiency system (HGPRT-).
376 CO--CH=N2 I
NH I CH2
~ DGE O - C H 2 - C H 3
;
R ~ DGA~NH2
L
I
co-R
DGI~NH_NH2
Fig. 1. S t r v c t u r e o f t h e 3 d i a z o a c e t y l g l y c i n e d e r i v a t i v e s .
DGE
DGA ~u z :::) O
DGI
2000
1500
N M.
~"
1000
-~ ~
sap
-~ Q ~,
~
3
5
~
3
7 EXPRESSION
5 TIME
7
3
S
~
IDAYS)
Fig. 2. E x p r e s s i o n t i m e of T G - r e s i s t a n t p h e n o t y p e . M u t a n t s p e r m i l l i o n viable cells axe p l o t t e d a g a i n s t e x p r e s s i o n t i m e in d a y s a f t e r t h e t r e a t m e n t w i t h 5 m M ( e ) a n d 10 m M (o) D G A , D G E a n d D G H . P o i n t s r e p r e s e n t the m e a n v a l u e s f r o m 2 i n d e p e n d e n t E x p t s . V e r t i c a l b a r s i n d i c a t e s t a n d a r d e r r o r s of the m e a n s (controls have been subtracted).
-: b) ~ ~
2000.
~,~ z ~ ~0o ~. ~~
~
~.
~.
A
I
25 8 DOSE/IO ~ ~
-
a)
I
10
Fig. 3. M u t a t i o n f r e q u e n c y (a) ~ d s u ~ i v ~ (b) a f t e r t r e a t m e n t w i t h d i ~ o a c e t y l ~ y c i n e d e ~ v a t i v e s D G A , D G E a n d D G H , as a ~ u n c t i o n o f t h e c o n c e n t r a t i o n . P o ~ t s ~ e the a v e r a g e f~om 4 i n d e p e n d e n t R x p t s . , a n d v e r t i c ~ b a r s r e p r e ~ n t the s t ~ d ~ d e r r o r s of the m e a n s ( c o n t r o l s h a v e b e e n s u b t r a c t e d ) , e , D G A ; a, D G E : ~, D G H .
377 Material and m e t h o d s Cell culture V79 Chinese hamster cells, originally obtained from Prof. Abbondandolo (Laboratorio di Mutagenesi e Differenziamento, CNR, Pisa) were grown in Dulbecco's minimal essential medium (DMEM), supplemented with 10% foetal calf serum (Microbiological Associates), penicillin at 100 IU/ml and streptomycin {Squibb) at 100 pg/ml, and were maintained in a humidified 5% CO: atmosphere at 37°C. Trypsin {1 : 250 Difco) at 0.25% in Dulbecco's phosphate-buffered saline (D-PBS) was routinely used for subculture. A clone of V79 cells, designated V79/D4, selected in our laboratory for its high plating efficiency and its low spontaneous mutation rate, was used throughout this work. Chemicals DGA, DGE and DGH (Fig. 1) were synthesized and kindly supplied by Prof. L. Baldini, Istituto di Farmacologia, Universit~ di Trieste. The 2-aminomercaptopurine (6-thioguanine, TG) was purchased from Sigma. Cell treatment V 7 9 / D 4 cells (2.5--3 × 106) were plated in 50-mm diameter dishes and incubated for 24 h at 37°C before the addition of the treatment solutions. The culture medium was replaced with fresh medium 1 h before the treatment. Cells were incubated for 1 h in 2.5, 5 and 10 mM solutions of each comp o u n d in medium without serum. Untreated controls were included in each experiment. At the end of the treatment, all the cultures were washed 4 times with medium without FBS. Cell survival and mutagenesis assay After mutagenic treatment, cells were detached from each dish with trypsin and the density of the resulting single~ell suspension was estimated in a haemocytometer. 5 × l 0 s cells were seeded in non-selective medium to propagate the culture from each expression time to the next (Abbondandolo et al., 1976). For the determination of the induced toxicity, 2 × 10: cells/50-mm dish, and for mutagenesis 2 × 10 s cells/100-mm dish, were plated. 1 h after the plating, 6-TG was added to the mutation plates at the final concentration of 4 pg/ml. To achieve maximal recovery of mutants, a complete expression time curve was constructed for 5 and 10 mM DGA, DGE and DGH. In practice, 5 replicates were treated with 6-TG at each of the 4 expressiontime points ranging from 0 to 7 days after treatment. Both survival and mutation plates were stained with 1% methylene blue 7 days after the plating, and colonies of more than a b o u t 50 cells were scored macroscopically. Results
The treatment of V 7 9 / D 4 cells with 2.5, 5 and 10 mM DGA, DGE or D G H induced a significantly higher number of 6-TG-resistant mutants than untreated controls, which never exceeded 30 mutants per 106 viable ceils.
378 Fig. 2 shows the frequency of 6-TG-resistant clones when the selective agent was added at various times after treatment with diazoacetylglycine derivatives. It is evident that a 5-day period was necessary for the expression of the maximal number of mutants. This is presumably the time necessary for the decay of pre-existing H G P R T enzyme and m R N A , which would occur through normal turnover mechanisms as well as dilution by cell division (Van Zeeland and Simons, 1976). In Fig. 3 the mutation frequency at 5
379
DGA >~ DGE > DGH. Furthermore, in the HGPRT system, the DGH is much less effective than the other derivatives, perhaps indicating a lower reactivity of the molecule carrying the hydrazide group. Acknowledgements We are grateful to Prof. Carlo Monti-Bragadin for useful discussions and to Prof. A. Abbondandolo (Laboratorio di Mutagenesi e Differenziamento, CNR, Pisa) for V79 cells. The technical assistance of Mrs. Presti and Mr. Gamboz is acknowledged. References Abbondandolo0 A., S. Bonatti, C. Colella, G. Corti, F. Matteucci, A. Mazzacaro and G. Rainaldi (1976) A comparative study of different e x p e r i m e n t a l protocol s for mutagenesis assays with 8-azaguanine resistance system in Chinese hamster cultured ceils, Mutation Res., 37, 293--306. Baldini, L., and G. Brambilla (1966) Antineoplastic activity of diazoacetylglycine derivatives, Cancer Res., 26, 1754--1758. Banfi, E., M. Tamaro, B. Pani and C. Monti-Bragadin (1974) Mechanism of the mutagenic activity of diazoacetylglycine derivatives, Boil. Ist. Sieroter. Milanese0 53, 632--635. Brambilla, G., S. Parodi, M. Cavanna and L. Baldini (1970) The immunodepressive activity of N-diazoacetylglyctne amide in some transplantation systems, Transplantation, 10, 100--105. Brambilla, G., M. Cavanna, S. Parodi and L. Baldini (1972) I n d u c t i o n of t umors in n e w b o r n and a dul t Swiss mice by N-diazoacetylglycine amide, Eur. J. Cancer, 8, 127--129. Gixaldi, T., and L. Baldini (1973) Mechanism of inhibition of DNA synthesis in Ehrlich ascites t u m o u r ceils by diazoacetylglycine amide, Biochem. Pharmacol., 22, 1793--1799. Monti-Bragadin, C., M. Tama~o and E. Banff (1974) Mutagenic activity of diazoacetylglycine derivatives, An timicrob. Agents Chemother., 6, 655--656. Parodi, S., C. Bolognesi, M. Cavanna, R.L. Pollack, L. Santi and G. Brambilla (1977a) Damage and repair of DNA in cultured m a m m a l i a n ceils with N-diazoacetylglycine amide, Cancer Res., 37, 4460---4466. Parodi, S., M. Picca, C. Bolognesi, M. Cavanna, P. Carlo, R. Finollo and G. Brambilla (1977b) A l k y l a t i o n of 7-(4-nitrobenzyl)pyridine by four N-diazoacetyl-derivatives of amino acids, Lack of fine correlation with the unscheduled DNA synthesis induced in mouse ceils "in vi t ro" , Pharmacol. Res. Commun.. 9,621-631. Van Zeeland, A.A., and J.W.I.M. Simons (1976) Linear dose--response relationships after prolonged expression times in V79 Chinese hamster ceils, Mutation Res., 35, 129--138.