- Email: [email protected]
Comparative mutagenicity of 4 DNA-intercalating agents in L5178Y mouse lymphoma cells
Mutation Research, 102 (1982) 447-455
447
Elsevier BiomedicalPress
Comparative mutagenicity of 4 DNA-intercalating agents in L5178Y mouse lymphoma cells Andrea M. Rogers and Kenneth C. Back Air Force Aerospace Medical Research Laboratory, Wright-Patterson Air Force Base, OH 45433 (U.S.A.)
(Received7 December 1981) (Revisionreceived15 March 1982) (Accepted 15 April 1982) Summary The mutagenicity of 4 known intercalating agents acridine orange (AO), quinacrine mustard (QM), proflavin (PF) and ethidium bromide (EB) has been investigated in L5178Y mouse lymphoma cells. Methyl methanesulfonate (MMS) was used as a positive control in these studies. AO, QM and PF induced mutation in the excess thymidine- and thioguanine-selective systems. These 3 compounds were negative in the ouabain- and cytosine-arabinoside-selective systems while EB was positive only in the cytosine arabinoside system. It would appear that the EB-induced mutagenesis is different from that of AO, QM and PF though all are intercalating agents. Since the molecular origin of cytosine arabinoside mutants is unknown, further interpretation of the EB results is not possible.
DNA-intercalating agents are generally recognized to be potent frameshift mutagens in bacterial systems (Ames et al., 1975). Studies with proflavin (PF) in bacteria have shown that PF is a direct-acting frameshift mutagen (Speck and Rosenkranz, 1980). In the dark, PF is a frameshift mutagen for S a l m o n e l l a t y p h i m u r i u m strains TA1537 and TA98. In the presence of microsomal enzymes, additional frameshift activity is seen in a strong positive response of the TA1538 strain. Exposure of bacteria to proflavin in the presence of visible light resulted in a positive response in strain TA1535, indicating base-pair substitution mutations (Speck and Rosenkranz, 1980). This is consistent with the observation that PF is a photogenerator of singlet oxygen (Ito, 1978)~ Acridine orange (AO) is positive in mutagenicity studies with Salmonella tester strains in the presence of metabolic activation (McCann et al., 1975). Ethidium bromide (EB), a trypanocidal drug, is widely used as a model compound in studies on the interaction between DNA and intercalating agents (Waring, 1970). In a Salmonella strain which detects frameshift mutagens, EB in the presence of an activating system is strongly positive (Mattern, 1976; McGregor and 0165-1218/82/0000-0000/$02.75 © ElsevierBiomedicalPress
448
( C H 3 ) 2 N ~ N
(CH3)2
H 2 N ' ~ ~ N H 2
Acridine Orange
~
NH2
Proflavine
c"3°'~~ ~cl ~.2c.2cl
r
Ethidium Bromide
2H20
H-N-CH~CH~CH,~CH~ I ~ ~ ~ ~/N CH3 / CH2CH2CI Quinacrine Mustard
Fig. 1. Structural comparison of DNA-intercalating agents.
Johnson, 1977). It did not cause base-pair substitutions and was negative in the absence of an activating system. In yeast, EB has been shown to cause 'petite' mutations and fragmentation of the mitochondrial DNA (Mattick and Nagley, 1977). In mammalian cells, intercalating agents are clastogenic (Ostertag and Kersten, 1965; Shaw, 1970; Hsu et al., 1977). A number of studies have been initiated to determine their potential to cause chromosomal aberrations both in established cell lines and in human lymphocytes (Kihlman et al., 1977). The induction of sister-chromatid exchange (SCE) by DNA-intercalating agents has also been examined in previous work. Most intercalating agents have been shown to increase the frequency of SCEs in human lymphocytes (Solomon and Bobrow, 1975; Crossen, 1979) and in Chinese hamster ovary (CHO) cells, an established cell line (Raj and Heddle, 1980; Perry and Evans, 1975). SCEs have been induced in mammalian cells by PF (Kato, 1974) and QM (Solomon and Bobrow, 1975; Perry and Evans, 1975). AO has been shown to cause mutation in the thymidine kinase selective system of L5178Y (TK ÷ / - ) mouse lymphoma cells (Amacher et al., 1979). This report is a systematic study of the mutagenicity of 4 intercalating agents in the L5178Y mammalian cell line. The structures of acridine orange, proflavin, quinacrine mustard and ethidium bromide are shown in Fig. I. Mutation studies with the alkylating agent methyl methanesulfonate (MMS) were used as a positive control. Materials and methods
The routine methods for maintenance of L5178¥ cells and the soft agar cloning technique were as described elsewhere (Cole and Arlett, 1976), except that McCoy's
449 5A medium was used instead of Fischer's medium. L5178Y were originally obtained from Dr. C.F. Arlett, MRC Cell Mutation Unit, Brighton, U.K, They were routinely screened for PPLO contamination. For range-finding toxicity experiments, L5178Y cells were treated for 24 h with doses of AO, PF, QM, and MMS. A treatment time of 48 h was used for EB. No induced mutation after 24 h treatment was noted for EB. All treatments were carried out in the dark since it has been shown that for at least 1 agent (PF), different results are obtained in the light (Speck and Rosenkranz, 1980). At the end of the treatment period, the cells were centrifuged, washed in McCoy's 5A medium (supplemented with penicillin, streptomycin, sodium pyruvate and 10% horse serum) and resuspended in McCoy's 5A medium (containing supplements + 20% horse serum). McCoy's 5A and horse sera were obtained from GibcoBiocult. Penicillin, streptomycin and sodium pyruvate were obtained from Sigma Ltd. The cells were then plated for survival in soft agar. The dose-response curve resulting from these experiments was used to determine suitable doses for the mutation experiments. 4 doses of AO, PF, QM and EB were examined in triplicate mutation experiments using a population of 107 cells at each dose level. 3 doses of MMS were examined in the same way. While evidence of some toxicity was desirable, survival rates of less than 40% were not deemed acceptable for mutation experiments. At the end of a 24-h or 48-h treatment, cells were centrifuged, washed and resuspended as in the toxicity experiments outlined above. The resuspended cells were plated at 0-h expression time and at 24-h intervals thereafter in selective medium for determination of mutation frequency and in nonselective medium for determination of survival. 4 selective agents, excess thymidine (TdR), ouabain (Oua), thioguanine (TG) and cytosine arabinoside (Ara-C) were used in each experiment. All selective agents were obtained from Sigma Ltd. and prepared as described in Cole and Arlett (1976) and Rogers et al. (1980). Maximum expression times were 24-48 h for Oua, 48-72 h for TdR and 144-192h for TG and Ara-C. Similar expression times were obtained in previous studies with these cells (Cole and Arlett, 1976; Rogers et al., 1980). Concentrations of the selective agents were as follows: 10-3M ouabain, 1.65 X 10 -3 M thymidine, 1.8 × 10 -4 M thioguanine and 10 -6 M cytosine arabinoside. Acridine orange, proflavin monohydrochloride, quinacrine mustard dihydrochloride and ethidium bromide were obtained from Sigma Ltd. MMS was obtained from Aldrich.
Results
Toxicity The toxicity for each compound is shown in Table 1. Similar results for AO treatment of L5178Y ceils were obtained by Amacher et al. (1979) after a 4-h treatment.
450 TABLE 1 SURVIVAL IN SOFT AGAR OF L5178Y CELLS Compound
Acridine orange (AO) b
Dose (raM X 10 -4)
Survivala (%)
1.5 7.5 11.2 15.0
98 +-0.8 94+--0.5 89+-0.8 66+2.7
0.2 0.9 1.8 3.7
98 +-0.6 86 +-0.9 77 + 1.2 47-+ 1.2
Proflavin (PF) b
4.0 10.0 12.0 16.0
96+- 1.3 86--+0.6 67+- 1.1 62+-0.5
Ethidium bromide (EB) ¢
2.5 7.6 12.7 25.4
83 +- 1.0 55+-0.9 66+- 1.5 31 -+2.8
240.0 360.0 480.0
94 +-0.6 77 -+0.7 58+-2.2
Quinacrine mustard (QM) b
Methyl methanesulfonate (MMS) b
a Mean of 3 Expts. b 24-h treatment in suspension. 48-h treatment in suspension.
Mutation AO, PF, Q M a n d M M S i n d u c e d t h y m i d i n e - a n d thioguanine-resistant variants. D o s e - r e s p o n s e curves (Figs. 2 a n d 3, T a b l e 2) were o b t a i n e d for these selective systems. I n d u c e d m u t a t i o n frequencies were calculated using the m e t h o d of Arlett a n d H a r c o u r t (1972). This . m e t h o d subtracts the s p o n t a n e o u s m u t a t i o n frequency f r o m the total m u t a t i o n frequency observed, hence providing the i n d u c e d m u t a t i o n frequency. S p o n t a n e o u s m u t a t i o n frequency to t h y m i d i n e resistance varied from 1.82 x 10 - 6 to 9.16 × 10 - 6 per survivor. The m e a n s p o n t a n e o u s m u t a t i o n frequency f r o m 12 experiments was 5.55 X 10 -6 per survivor. S p o n t a n e o u s m u t a t i o n frequency to t h i o g u a n i n e resistance was in the range 0.84 X 10 - 6 to 4.38 × 10 -6 per survivor. T h e m e a n s p o n t a n e o u s m u t a t i o n frequency from 12 experiments was 1.93 X 10 - 6 per survivor. AO, PF, Q M a n d M M S i n d u c e d n o significant m u t a t i o n in the o u a b a i n - or cytosine-arabinoside-seleetive systems. E t h i d i u m b r o m i d e significantly increased m u t a t i o n only in the c y t o s i n e - a r a b i n o side-selective system (Fig. 4). The results o b t a i n e d i n this selective system for EB m u t a g e n e s i s showed m u c h greater variability than n o r m a l between experiments. The variability does n o t seem to be a characteristic of the selective system since results
451
16
14 o x
12
._>
10 8 O
3
E
6
"O @
g m
4
2
0
i
i
6
8
1'0 1'2 14
16 1'11
Dose 10-4 mM
Fig. 2. Mutation induction in the excess thymidine-selective system by acridine orange ( A ) , proflavin ( • ) , a n d quinacrine mustard (0). Treatment time was 24 h for each compound. Mean of 3 Expts. with standard error.
obtained for induction of Ara-C r mutants after ethyl methanesulfonate (EMS) and 2-acetyl aminofluorine treatment show normal variability (Rogers et al., 1980). The large standard errors may be an expression of the increased toxicity of EB over EMS, or may be influenced by the difference in treatment times (2 h for EMS and 48 h for EB). There was no significant mutation in the ouabain-, thymidine- or
TABLE 2 M U T A T I O N ' I N D U C T I O N BY MMS a Dose ( × 10 - 2 raM)
Induced mutants per survivor b ( × 10 - 5 ) Thymidine Thioguanine
2.4 3.6 4.8
16.3 ±0.49 24.8-----0.44 28.8 -4-0.70
a 24-h treatment of L5178Y. b Mean of 3 Expts, with standard error.
12.7--+0.11 19.2±0.10 26.3 -4-0.08
452
80-
/
70-
o x
60
~
50
1
?, D.
4O
c o
3 E
30'
"0
S =) -o c
20-
10. ar 0
2
4
-6
~__ 8
10
12
1'4
i6
i8
Dose x 10 -4 m M
Fig. 3. Mutation induction in the thioguanine selective system by acridine orange (&), proflavin ( 1 ) , and quinacrine mustard (Q). Treatment time was 24 h for each compound. Mean of 3 Expts. with standard error.
o x at
0.4
O > ag
0.3 e~
z <
0.2
IE ~
0.1
0
z ...1.
J
2
i
4
6
8
lO
12 DOSE
14
16
xl0 -4
18 mM
20
22
24
26
i
i
28
I
|
30
EB
Fig. 4. Mutation induction in the cytosine arabinoside-selective system by ethidium bromide (48 h treatment). Mean of 3 Expts. with standard error.
453 thioguanine-selective system. A very low spontaneous mutation frequency for resistance to Ara-C was observed. The mutation frequency was 2.40 × 10 -8 per survivor which is comparable to that previously reported for these cells (Rogers et al., 1980).
Discussion
Studies with LS178Y mouse lymphoma cells were initiated to establish the response of these cells to a group of DNA-intercalating agents. 3 of the agents, AO, QM, and PF had mutation-induction patterns similar to that of the alkylating agent M M S which served as the positive control. The 4th intercalating agent, EB, had a quite different mutation-induction pattern. AO, QM, PF and MMS induced mutations in the excess thymidine and thioguanine systems. If mutation induction is compared through survivals (i.e. equitoxicity) rather than dose (Table3), the intercalating agents induced fewer mutants in the excess thymidine-selective system than MMS. In the thioguanine system, Q M was the most mutagenic compound followed by MMS, acridine orange and proflavin. The results we obtained with MMS are in agreement with results obtained for 20-h treatment of LS178Y cells with MMS by Cole and Arlett (1978). They also reported a lack of induction of Oua resistance for MMS. The DNA-intercalating agents have been shown to be frameshift mutagens in bacteria. It is well established that Oua resistance arises by alterations in an essential enzyme, the N a + / K + ATPase of the plasma membrane. It is probable that mutations which radically alter the properties or lead to a complete functional loss of this enzyme would be lethal. The frameshift mutations seen in bacteria induced by DNA-intercalating agents would fall into this category. It may be inferred that mutagens which do induce Oua resistance, produce mainly base-pair substitutions (Leever and Seegmiller, 1976; Arlett et al., 1975). The results of this study could indicate that AO, PF and QM produce frameshift mutations with very little production of base-pair substitution mutation. The results for EB are difficult to interpret. Our results confirm studies carried
TABLE 3 COMPARISON OF INDUCED MUTATION FREQUENCY AGAINST SURVIVALa Compound
AO PF . QM MMS
Survival
67 67 67 67
Induced mutants per survivor (× 10-5) Excess thymidine
Thioguanine
2.62 ± 0.84 2.95 -- 0.56 10.38 b 26.80 b
2.43 ± 0.24 1.39± 0.03 51.10 b 22.75 b
a A survival rate of 67% was arbitrarily chosen to make the comparison in mutation frequencies. b Obtained by interpolation.
454 out with another preparation of EB from a different source (Rogers, 1978; Rogers et al., 1980). EB induces only Ara-C-resistant mutants. However, the molecular origin of the Ara-C-resistant mutations is not known. It is known that EB produces 2 types of mutant that are morphologically distinct (Rogers et al., 1980). Ara-C-resistant cells in class I have no detectable deoxycytidine kinase activity, while resistant cells in class II have approximately 50% of the deoxycytidine kinase activity of wild-type cells. The enzymatic defect in these latter cells is obscure but the phenotypic properties probably result from subtle alterations in the pool of deoxycytidine nucleotides. Further interpretation of the EB results will only be possible when the molecular nature of the Ara-C mutants is known. Thus, a systematic study of 4 DNA-intercalating agents has shown that all 4 are direct-acting mutagens in L5178Y mouse lymphoma cells. AO, Q M and PF induce mutation in the excess thymidine- and thioguanine-selective systems and QM is clearly the most mutagenic of the compounds at these levels. It is not possible to characterize the precise molecular nature of these mutations since there is no direct evidence for the genetic changes accompanying resistance to the selective agents. However, the lack of increased mutation in the Oua system indicates that the intercalating agents probably do not cause base-pair substitutions. A number of studies in bacteria have shown that intercalating agents mainly produce frameshift mutations (Ames and Whitfield, 1966; Brusick and Zeiger, 1972; McCann et al., 1975). The data could be interpreted to indicate that AO, PF and QM cause frameshift mutations in these cells. Further, EB produces mutations of a different character. Since the molecular origin of Ara-C mutants is unknown, these mutations m a y well be of a frameshift nature. However, even if this is the case, the molecular mechanism underlying EB mutagenesis is clearly of a different nature than AO-, QM- and PF-induced mutagenesis.
Acknowledgements A.M. Rogers was .in receipt of a National Academy of Sciences, National Research Council Research Associateship. We are indebted to Dr. C.F. Arlett, M R C Cell Mutation Unit, Brighton, U.K. and Dr. M.E. Andersen, A F A M R L / T H T , Wright-Patterson Air Force Base, Ohio for m a n y helpful comments in the preparation of this report.
References Amacher, D.E., S. Paillet and V.A. Ray(1979) Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, Application to genetic toxicologicaltesting, Mutation Res., 64, 391-406. Ames, B.N., and H.J. Whitfield Jr. (1966) Frameshift mutagenesis in Salmonella, Cold Spring Harbor Symp. Quant. Biol., 31,221-225. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens with the Salmonella/mammalian microsome mutagenicity test, Mutation Res., 31,377-364. Arlett, C.F., and S.A. Harcourt (1972) Expression time and spontaneous mutability in the estimation of
455 induced mutation frequency following treatment of Chinese hamster cells by ultraviolet light, Mutation Res., 16, 301-306. Brusick, D.J., and E. Zeiger (1972) A comparison of chemically induced reversion patterns of Salmonella typhimurium and Saccharomyces cerevisiae mutants using in vitro test plates, Mutation Res., 14, 271-275. Cole, J., and C.F. Arlett (1976) Ethyl methane-sulphonate mutagenesis with L5178Y mouse lymphoma cells: a comparison of ouabain, thioguanine and excess thymidine resistance, Mutation Res., 34, 507-526. Cole, J., and C.F. Arlett (1978) Methyl methane-sulphonate mutagenesis in L5178Y mouse lymphoma cells, Mutation Res., 50, 111-120. Crossen, P. (1979) The effect of acridine compounds on sister-chromatid exchange formation in cultured human lymphocytes, Mutation Res., 68, 295-299. Hsu, T.C., C.J. Collie, A.F. Lusby and D.A. Johnston (1977) Cytogenetic assays of chemical clastogens using mammalian cells in culture, Mutation Res., 45, 233-247. Ito, T. (1978) Cellular and subcellular mechanisms of photodynamic action: The IO 2 hypothesis as a driving force in recent research, Photochem. Photobiol., 28, 493-508. Kato, H. (1974) Induction of sister chromatid exchanges by chemical mutagens and its possible relation to DNA repair, Exp. Cell. Res., 85, 239-247. Kihlman, B.A., S. Sturelid, F. Palitti and A. Becchetti (1977) Effects of caffeine, an inhibitor of post-replication repair in mammalian cells, on the frequencies of chromosomal aberrations and sister chromatid exchanges induced by mutagenic agents, Mutation Res., 46, 130-131. Mattern, I.E. (1976) Mutagenicity of ethidium bromide after metabolic activation in vitro, Mutation Res., 38, 120. Mattick, J.S., and P. Nagley (1977) Comparative studies of the effects of acridines and other petite inducing drugs on the mitochondrial genome of Saccharomyces cerevisiae, Mol. Gen. Genet., 152, 267-276. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 4135-4139. McGregor, J.T., and I.J. Johnson (1977) In vitro metabolic activation of ethidium bromide and other phenanthridium compounds, Mutagenic activity in Salmonella typhimurium, Mutation Res., 48, 103-108. Ostertag, W., and W. Kersten (1965) The action of proflavin and actinomycin D in causing chromosome breakage in human cells, Exp. Cell. Res., 39, 296-300. Perry, P., and H.J. Evans (1975) Cytological detection of mutagen/carcinogen exposure by sister chromatid exchange, Nature (London), 258, 121-125. Raj, A.S., and J.A. Heddle (1980) Simultaneous detection of chromosome aberrations and sister chromatid exchanges, Experience with DNA intercalating agents, Mutation Res., 78, 253-260. Rogers, A.M. '(1978) Mutagenicity testing with mouse cells a n d bacteria, D. Phil. Thesis, University of Sussex, Brighton. Rogers, A.M., R. Hill, A.R. Lehmann, C.F. Arlett and V.W. Burns (1980) The induction and characterization of mouse lymphoma L5178Y cell lines resistant to l-fl-D-arabinofuranosylcytosine, Mutation Res., 69, 139-148. Shaw, M.W. (1970) Human chromosome damage by chemical agents, Annu. Rev. Med., 409-432. Solomon, E., and M. Bobrow (1975) Sister chromatid exchanges - - a sensitive assay of agents damaging human chromosomes, Mutation Res., 30, 273-278. Speck, W.T., and H.S. Rosenkranz (1980) Proflavin: an unusual mutagen, Mutation Res., 77, 37-43. Waring, M. (1970) Variation of the supercoils in closed circular DNA by binding of antibiotics and drugs: evidence for molecular models involving intercalation, J. Mol. Biol., 54, 247-279.
447
Elsevier BiomedicalPress
Comparative mutagenicity of 4 DNA-intercalating agents in L5178Y mouse lymphoma cells Andrea M. Rogers and Kenneth C. Back Air Force Aerospace Medical Research Laboratory, Wright-Patterson Air Force Base, OH 45433 (U.S.A.)
(Received7 December 1981) (Revisionreceived15 March 1982) (Accepted 15 April 1982) Summary The mutagenicity of 4 known intercalating agents acridine orange (AO), quinacrine mustard (QM), proflavin (PF) and ethidium bromide (EB) has been investigated in L5178Y mouse lymphoma cells. Methyl methanesulfonate (MMS) was used as a positive control in these studies. AO, QM and PF induced mutation in the excess thymidine- and thioguanine-selective systems. These 3 compounds were negative in the ouabain- and cytosine-arabinoside-selective systems while EB was positive only in the cytosine arabinoside system. It would appear that the EB-induced mutagenesis is different from that of AO, QM and PF though all are intercalating agents. Since the molecular origin of cytosine arabinoside mutants is unknown, further interpretation of the EB results is not possible.
DNA-intercalating agents are generally recognized to be potent frameshift mutagens in bacterial systems (Ames et al., 1975). Studies with proflavin (PF) in bacteria have shown that PF is a direct-acting frameshift mutagen (Speck and Rosenkranz, 1980). In the dark, PF is a frameshift mutagen for S a l m o n e l l a t y p h i m u r i u m strains TA1537 and TA98. In the presence of microsomal enzymes, additional frameshift activity is seen in a strong positive response of the TA1538 strain. Exposure of bacteria to proflavin in the presence of visible light resulted in a positive response in strain TA1535, indicating base-pair substitution mutations (Speck and Rosenkranz, 1980). This is consistent with the observation that PF is a photogenerator of singlet oxygen (Ito, 1978)~ Acridine orange (AO) is positive in mutagenicity studies with Salmonella tester strains in the presence of metabolic activation (McCann et al., 1975). Ethidium bromide (EB), a trypanocidal drug, is widely used as a model compound in studies on the interaction between DNA and intercalating agents (Waring, 1970). In a Salmonella strain which detects frameshift mutagens, EB in the presence of an activating system is strongly positive (Mattern, 1976; McGregor and 0165-1218/82/0000-0000/$02.75 © ElsevierBiomedicalPress
448
( C H 3 ) 2 N ~ N
(CH3)2
H 2 N ' ~ ~ N H 2
Acridine Orange
~
NH2
Proflavine
c"3°'~~ ~cl ~.2c.2cl
r
Ethidium Bromide
2H20
H-N-CH~CH~CH,~CH~ I ~ ~ ~ ~/N CH3 / CH2CH2CI Quinacrine Mustard
Fig. 1. Structural comparison of DNA-intercalating agents.
Johnson, 1977). It did not cause base-pair substitutions and was negative in the absence of an activating system. In yeast, EB has been shown to cause 'petite' mutations and fragmentation of the mitochondrial DNA (Mattick and Nagley, 1977). In mammalian cells, intercalating agents are clastogenic (Ostertag and Kersten, 1965; Shaw, 1970; Hsu et al., 1977). A number of studies have been initiated to determine their potential to cause chromosomal aberrations both in established cell lines and in human lymphocytes (Kihlman et al., 1977). The induction of sister-chromatid exchange (SCE) by DNA-intercalating agents has also been examined in previous work. Most intercalating agents have been shown to increase the frequency of SCEs in human lymphocytes (Solomon and Bobrow, 1975; Crossen, 1979) and in Chinese hamster ovary (CHO) cells, an established cell line (Raj and Heddle, 1980; Perry and Evans, 1975). SCEs have been induced in mammalian cells by PF (Kato, 1974) and QM (Solomon and Bobrow, 1975; Perry and Evans, 1975). AO has been shown to cause mutation in the thymidine kinase selective system of L5178Y (TK ÷ / - ) mouse lymphoma cells (Amacher et al., 1979). This report is a systematic study of the mutagenicity of 4 intercalating agents in the L5178Y mammalian cell line. The structures of acridine orange, proflavin, quinacrine mustard and ethidium bromide are shown in Fig. I. Mutation studies with the alkylating agent methyl methanesulfonate (MMS) were used as a positive control. Materials and methods
The routine methods for maintenance of L5178¥ cells and the soft agar cloning technique were as described elsewhere (Cole and Arlett, 1976), except that McCoy's
449 5A medium was used instead of Fischer's medium. L5178Y were originally obtained from Dr. C.F. Arlett, MRC Cell Mutation Unit, Brighton, U.K, They were routinely screened for PPLO contamination. For range-finding toxicity experiments, L5178Y cells were treated for 24 h with doses of AO, PF, QM, and MMS. A treatment time of 48 h was used for EB. No induced mutation after 24 h treatment was noted for EB. All treatments were carried out in the dark since it has been shown that for at least 1 agent (PF), different results are obtained in the light (Speck and Rosenkranz, 1980). At the end of the treatment period, the cells were centrifuged, washed in McCoy's 5A medium (supplemented with penicillin, streptomycin, sodium pyruvate and 10% horse serum) and resuspended in McCoy's 5A medium (containing supplements + 20% horse serum). McCoy's 5A and horse sera were obtained from GibcoBiocult. Penicillin, streptomycin and sodium pyruvate were obtained from Sigma Ltd. The cells were then plated for survival in soft agar. The dose-response curve resulting from these experiments was used to determine suitable doses for the mutation experiments. 4 doses of AO, PF, QM and EB were examined in triplicate mutation experiments using a population of 107 cells at each dose level. 3 doses of MMS were examined in the same way. While evidence of some toxicity was desirable, survival rates of less than 40% were not deemed acceptable for mutation experiments. At the end of a 24-h or 48-h treatment, cells were centrifuged, washed and resuspended as in the toxicity experiments outlined above. The resuspended cells were plated at 0-h expression time and at 24-h intervals thereafter in selective medium for determination of mutation frequency and in nonselective medium for determination of survival. 4 selective agents, excess thymidine (TdR), ouabain (Oua), thioguanine (TG) and cytosine arabinoside (Ara-C) were used in each experiment. All selective agents were obtained from Sigma Ltd. and prepared as described in Cole and Arlett (1976) and Rogers et al. (1980). Maximum expression times were 24-48 h for Oua, 48-72 h for TdR and 144-192h for TG and Ara-C. Similar expression times were obtained in previous studies with these cells (Cole and Arlett, 1976; Rogers et al., 1980). Concentrations of the selective agents were as follows: 10-3M ouabain, 1.65 X 10 -3 M thymidine, 1.8 × 10 -4 M thioguanine and 10 -6 M cytosine arabinoside. Acridine orange, proflavin monohydrochloride, quinacrine mustard dihydrochloride and ethidium bromide were obtained from Sigma Ltd. MMS was obtained from Aldrich.
Results
Toxicity The toxicity for each compound is shown in Table 1. Similar results for AO treatment of L5178Y ceils were obtained by Amacher et al. (1979) after a 4-h treatment.
450 TABLE 1 SURVIVAL IN SOFT AGAR OF L5178Y CELLS Compound
Acridine orange (AO) b
Dose (raM X 10 -4)
Survivala (%)
1.5 7.5 11.2 15.0
98 +-0.8 94+--0.5 89+-0.8 66+2.7
0.2 0.9 1.8 3.7
98 +-0.6 86 +-0.9 77 + 1.2 47-+ 1.2
Proflavin (PF) b
4.0 10.0 12.0 16.0
96+- 1.3 86--+0.6 67+- 1.1 62+-0.5
Ethidium bromide (EB) ¢
2.5 7.6 12.7 25.4
83 +- 1.0 55+-0.9 66+- 1.5 31 -+2.8
240.0 360.0 480.0
94 +-0.6 77 -+0.7 58+-2.2
Quinacrine mustard (QM) b
Methyl methanesulfonate (MMS) b
a Mean of 3 Expts. b 24-h treatment in suspension. 48-h treatment in suspension.
Mutation AO, PF, Q M a n d M M S i n d u c e d t h y m i d i n e - a n d thioguanine-resistant variants. D o s e - r e s p o n s e curves (Figs. 2 a n d 3, T a b l e 2) were o b t a i n e d for these selective systems. I n d u c e d m u t a t i o n frequencies were calculated using the m e t h o d of Arlett a n d H a r c o u r t (1972). This . m e t h o d subtracts the s p o n t a n e o u s m u t a t i o n frequency f r o m the total m u t a t i o n frequency observed, hence providing the i n d u c e d m u t a t i o n frequency. S p o n t a n e o u s m u t a t i o n frequency to t h y m i d i n e resistance varied from 1.82 x 10 - 6 to 9.16 × 10 - 6 per survivor. The m e a n s p o n t a n e o u s m u t a t i o n frequency f r o m 12 experiments was 5.55 X 10 -6 per survivor. S p o n t a n e o u s m u t a t i o n frequency to t h i o g u a n i n e resistance was in the range 0.84 X 10 - 6 to 4.38 × 10 -6 per survivor. T h e m e a n s p o n t a n e o u s m u t a t i o n frequency from 12 experiments was 1.93 X 10 - 6 per survivor. AO, PF, Q M a n d M M S i n d u c e d n o significant m u t a t i o n in the o u a b a i n - or cytosine-arabinoside-seleetive systems. E t h i d i u m b r o m i d e significantly increased m u t a t i o n only in the c y t o s i n e - a r a b i n o side-selective system (Fig. 4). The results o b t a i n e d i n this selective system for EB m u t a g e n e s i s showed m u c h greater variability than n o r m a l between experiments. The variability does n o t seem to be a characteristic of the selective system since results
451
16
14 o x
12
._>
10 8 O
3
E
6
"O @
g m
4
2
0
i
i
6
8
1'0 1'2 14
16 1'11
Dose 10-4 mM
Fig. 2. Mutation induction in the excess thymidine-selective system by acridine orange ( A ) , proflavin ( • ) , a n d quinacrine mustard (0). Treatment time was 24 h for each compound. Mean of 3 Expts. with standard error.
obtained for induction of Ara-C r mutants after ethyl methanesulfonate (EMS) and 2-acetyl aminofluorine treatment show normal variability (Rogers et al., 1980). The large standard errors may be an expression of the increased toxicity of EB over EMS, or may be influenced by the difference in treatment times (2 h for EMS and 48 h for EB). There was no significant mutation in the ouabain-, thymidine- or
TABLE 2 M U T A T I O N ' I N D U C T I O N BY MMS a Dose ( × 10 - 2 raM)
Induced mutants per survivor b ( × 10 - 5 ) Thymidine Thioguanine
2.4 3.6 4.8
16.3 ±0.49 24.8-----0.44 28.8 -4-0.70
a 24-h treatment of L5178Y. b Mean of 3 Expts, with standard error.
12.7--+0.11 19.2±0.10 26.3 -4-0.08
452
80-
/
70-
o x
60
~
50
1
?, D.
4O
c o
3 E
30'
"0
S =) -o c
20-
10. ar 0
2
4
-6
~__ 8
10
12
1'4
i6
i8
Dose x 10 -4 m M
Fig. 3. Mutation induction in the thioguanine selective system by acridine orange (&), proflavin ( 1 ) , and quinacrine mustard (Q). Treatment time was 24 h for each compound. Mean of 3 Expts. with standard error.
o x at
0.4
O > ag
0.3 e~
z <
0.2
IE ~
0.1
0
z ...1.
J
2
i
4
6
8
lO
12 DOSE
14
16
xl0 -4
18 mM
20
22
24
26
i
i
28
I
|
30
EB
Fig. 4. Mutation induction in the cytosine arabinoside-selective system by ethidium bromide (48 h treatment). Mean of 3 Expts. with standard error.
453 thioguanine-selective system. A very low spontaneous mutation frequency for resistance to Ara-C was observed. The mutation frequency was 2.40 × 10 -8 per survivor which is comparable to that previously reported for these cells (Rogers et al., 1980).
Discussion
Studies with LS178Y mouse lymphoma cells were initiated to establish the response of these cells to a group of DNA-intercalating agents. 3 of the agents, AO, QM, and PF had mutation-induction patterns similar to that of the alkylating agent M M S which served as the positive control. The 4th intercalating agent, EB, had a quite different mutation-induction pattern. AO, QM, PF and MMS induced mutations in the excess thymidine and thioguanine systems. If mutation induction is compared through survivals (i.e. equitoxicity) rather than dose (Table3), the intercalating agents induced fewer mutants in the excess thymidine-selective system than MMS. In the thioguanine system, Q M was the most mutagenic compound followed by MMS, acridine orange and proflavin. The results we obtained with MMS are in agreement with results obtained for 20-h treatment of LS178Y cells with MMS by Cole and Arlett (1978). They also reported a lack of induction of Oua resistance for MMS. The DNA-intercalating agents have been shown to be frameshift mutagens in bacteria. It is well established that Oua resistance arises by alterations in an essential enzyme, the N a + / K + ATPase of the plasma membrane. It is probable that mutations which radically alter the properties or lead to a complete functional loss of this enzyme would be lethal. The frameshift mutations seen in bacteria induced by DNA-intercalating agents would fall into this category. It may be inferred that mutagens which do induce Oua resistance, produce mainly base-pair substitutions (Leever and Seegmiller, 1976; Arlett et al., 1975). The results of this study could indicate that AO, PF and QM produce frameshift mutations with very little production of base-pair substitution mutation. The results for EB are difficult to interpret. Our results confirm studies carried
TABLE 3 COMPARISON OF INDUCED MUTATION FREQUENCY AGAINST SURVIVALa Compound
AO PF . QM MMS
Survival
67 67 67 67
Induced mutants per survivor (× 10-5) Excess thymidine
Thioguanine
2.62 ± 0.84 2.95 -- 0.56 10.38 b 26.80 b
2.43 ± 0.24 1.39± 0.03 51.10 b 22.75 b
a A survival rate of 67% was arbitrarily chosen to make the comparison in mutation frequencies. b Obtained by interpolation.
454 out with another preparation of EB from a different source (Rogers, 1978; Rogers et al., 1980). EB induces only Ara-C-resistant mutants. However, the molecular origin of the Ara-C-resistant mutations is not known. It is known that EB produces 2 types of mutant that are morphologically distinct (Rogers et al., 1980). Ara-C-resistant cells in class I have no detectable deoxycytidine kinase activity, while resistant cells in class II have approximately 50% of the deoxycytidine kinase activity of wild-type cells. The enzymatic defect in these latter cells is obscure but the phenotypic properties probably result from subtle alterations in the pool of deoxycytidine nucleotides. Further interpretation of the EB results will only be possible when the molecular nature of the Ara-C mutants is known. Thus, a systematic study of 4 DNA-intercalating agents has shown that all 4 are direct-acting mutagens in L5178Y mouse lymphoma cells. AO, Q M and PF induce mutation in the excess thymidine- and thioguanine-selective systems and QM is clearly the most mutagenic of the compounds at these levels. It is not possible to characterize the precise molecular nature of these mutations since there is no direct evidence for the genetic changes accompanying resistance to the selective agents. However, the lack of increased mutation in the Oua system indicates that the intercalating agents probably do not cause base-pair substitutions. A number of studies in bacteria have shown that intercalating agents mainly produce frameshift mutations (Ames and Whitfield, 1966; Brusick and Zeiger, 1972; McCann et al., 1975). The data could be interpreted to indicate that AO, PF and QM cause frameshift mutations in these cells. Further, EB produces mutations of a different character. Since the molecular origin of Ara-C mutants is unknown, these mutations m a y well be of a frameshift nature. However, even if this is the case, the molecular mechanism underlying EB mutagenesis is clearly of a different nature than AO-, QM- and PF-induced mutagenesis.
Acknowledgements A.M. Rogers was .in receipt of a National Academy of Sciences, National Research Council Research Associateship. We are indebted to Dr. C.F. Arlett, M R C Cell Mutation Unit, Brighton, U.K. and Dr. M.E. Andersen, A F A M R L / T H T , Wright-Patterson Air Force Base, Ohio for m a n y helpful comments in the preparation of this report.
References Amacher, D.E., S. Paillet and V.A. Ray(1979) Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, Application to genetic toxicologicaltesting, Mutation Res., 64, 391-406. Ames, B.N., and H.J. Whitfield Jr. (1966) Frameshift mutagenesis in Salmonella, Cold Spring Harbor Symp. Quant. Biol., 31,221-225. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens with the Salmonella/mammalian microsome mutagenicity test, Mutation Res., 31,377-364. Arlett, C.F., and S.A. Harcourt (1972) Expression time and spontaneous mutability in the estimation of
455 induced mutation frequency following treatment of Chinese hamster cells by ultraviolet light, Mutation Res., 16, 301-306. Brusick, D.J., and E. Zeiger (1972) A comparison of chemically induced reversion patterns of Salmonella typhimurium and Saccharomyces cerevisiae mutants using in vitro test plates, Mutation Res., 14, 271-275. Cole, J., and C.F. Arlett (1976) Ethyl methane-sulphonate mutagenesis with L5178Y mouse lymphoma cells: a comparison of ouabain, thioguanine and excess thymidine resistance, Mutation Res., 34, 507-526. Cole, J., and C.F. Arlett (1978) Methyl methane-sulphonate mutagenesis in L5178Y mouse lymphoma cells, Mutation Res., 50, 111-120. Crossen, P. (1979) The effect of acridine compounds on sister-chromatid exchange formation in cultured human lymphocytes, Mutation Res., 68, 295-299. Hsu, T.C., C.J. Collie, A.F. Lusby and D.A. Johnston (1977) Cytogenetic assays of chemical clastogens using mammalian cells in culture, Mutation Res., 45, 233-247. Ito, T. (1978) Cellular and subcellular mechanisms of photodynamic action: The IO 2 hypothesis as a driving force in recent research, Photochem. Photobiol., 28, 493-508. Kato, H. (1974) Induction of sister chromatid exchanges by chemical mutagens and its possible relation to DNA repair, Exp. Cell. Res., 85, 239-247. Kihlman, B.A., S. Sturelid, F. Palitti and A. Becchetti (1977) Effects of caffeine, an inhibitor of post-replication repair in mammalian cells, on the frequencies of chromosomal aberrations and sister chromatid exchanges induced by mutagenic agents, Mutation Res., 46, 130-131. Mattern, I.E. (1976) Mutagenicity of ethidium bromide after metabolic activation in vitro, Mutation Res., 38, 120. Mattick, J.S., and P. Nagley (1977) Comparative studies of the effects of acridines and other petite inducing drugs on the mitochondrial genome of Saccharomyces cerevisiae, Mol. Gen. Genet., 152, 267-276. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 4135-4139. McGregor, J.T., and I.J. Johnson (1977) In vitro metabolic activation of ethidium bromide and other phenanthridium compounds, Mutagenic activity in Salmonella typhimurium, Mutation Res., 48, 103-108. Ostertag, W., and W. Kersten (1965) The action of proflavin and actinomycin D in causing chromosome breakage in human cells, Exp. Cell. Res., 39, 296-300. Perry, P., and H.J. Evans (1975) Cytological detection of mutagen/carcinogen exposure by sister chromatid exchange, Nature (London), 258, 121-125. Raj, A.S., and J.A. Heddle (1980) Simultaneous detection of chromosome aberrations and sister chromatid exchanges, Experience with DNA intercalating agents, Mutation Res., 78, 253-260. Rogers, A.M. '(1978) Mutagenicity testing with mouse cells a n d bacteria, D. Phil. Thesis, University of Sussex, Brighton. Rogers, A.M., R. Hill, A.R. Lehmann, C.F. Arlett and V.W. Burns (1980) The induction and characterization of mouse lymphoma L5178Y cell lines resistant to l-fl-D-arabinofuranosylcytosine, Mutation Res., 69, 139-148. Shaw, M.W. (1970) Human chromosome damage by chemical agents, Annu. Rev. Med., 409-432. Solomon, E., and M. Bobrow (1975) Sister chromatid exchanges - - a sensitive assay of agents damaging human chromosomes, Mutation Res., 30, 273-278. Speck, W.T., and H.S. Rosenkranz (1980) Proflavin: an unusual mutagen, Mutation Res., 77, 37-43. Waring, M. (1970) Variation of the supercoils in closed circular DNA by binding of antibiotics and drugs: evidence for molecular models involving intercalation, J. Mol. Biol., 54, 247-279.