- Email: [email protected]
mammalian microsome mutagenicity test
Mutation Research, 209 (1988) 57-62
57
Elsevier
MTRL 0138
Verapamil as a co-mutagen in the Salmonella/mammalian microsome mutagenicity test L y n n e t t e R. F e r g u s o n a n d Bruce C. B a g u l e y Cancer Research Laboratory, University of Auckland Medical School, Private Bag, Auckland (New Zealand) (Accepted 25 May 1988)
Keywords." Verapamil; Calcium-channel blocking; Anilinoacridines
Summary Verapamil is a calcium-channel blocking agent, commonly used for chronic treatment of heart conditions. We have previously demonstrated that verapamil acts as a co-mutagen in a bacterial mutagenicity test for some experimental anilinoacridine antitumour drugs. Within the anilinoacridines series there are several compounds which are apparently non-mutagenic (or very weak mutagens) in the absence of verapamil, but strong mutagens in its presence. We have now tested a wider range of materials for verapamil enhancement of mutagenicity, to include some of those to which persons on verapamil therapy might be exposed through life-style or occupation. Some verapamil enhancement of mutagenicity was seen with most mutagenic compounds including anticancer drugs, antiparasitic agents, one biological stain and one hair dye. A number of tricyclic antidepressants and biological stains were tested and found to be non-mutagenic. If these results extrapolate to mammalian cells, long-term verapamil therapy could potentially increase the effects of certain enironmental mutagens.
Verapamil is a calcium-channel blocking agent which is used clinically, particularly for the control of various heart complaints such as arrhythmia and angina pectoris (Reynolds and Prasad, 1982). Recommended doses of verapamil may reach 360 mg/day over extended time periods. Verapamil is generally considered to be relatively free from harmful side effects in comparison with other Correspondence: Dr. Lynnette R. Ferguson, Cancer Research Laboratory, University of Auckland Medical School, Private Bag, Auckland (New Zealand).
heart medication (Reynolds and Prasar, 1982). Verapamil is also notable in overcoming resistance to a variety of antibiotics and plant products in multidrug resistant mammalian cells (Tsuruo et al., 1981). In a preliminary study on a group of 3 anilinoacridines and 1 acridine (proflavine), we noted that mutagenicity in the Salmonella mammalian/microsome mutagenicity test could be enhanced, to varying degrees for the different compounds, by verapamil. We also found that the potency of those compounds whose mutagenicity
0165-7992/88/$ 03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
58
was most strongly affected by verapamil in bacteria, was greatly increased by verapamil in a multidrug resistant mammalian cell line (Baguley and Ferguson, 1986). If these effects of verapamil are restricted to a small group of experimental drugs to which the general public are not likely to be exposed, then the phenomenon may be only of theoretical interest. However, if the enhancements are seen with other types of compound in the environment, then they could have rather wider implications for persons on long term verapamil therapy. From our previous studies with anilinoacridine derivatives, it appeared that other strongly basic, hydrophobic molecules would be likely to show similar effects. In this study, we have investigated the ability of verapamil to enhance mutagenicity of
a range of materials, including hair dyes, biological stains, acridine derivatives, tricyclic antidepressants and anticancer drugs. Most of the compounds selected fit the above criteria. Structures of the compounds are described in Table 1, or in either the Merck Index or Reynolds and Prasad, 1982. Materials and methods
Compounds Verapamil was produced by Knoll A.G., Ludwigshafen (Germany). Anilinoacridines (Denny et al., 1982), nitracrine (Hrabowska et al., 1982), NSC 601 316 (Atwell et al., 1987), NSC 176 319 (Denny et al., 1979) and NSC 342 965 (Atwell et al., 1984) were synthesised in the Cancer Research
TABLE 1 M U T A G E N I C I T Y OF VARIOUS C O M P O U N D S IN 3 STRAINS OF S. typhimurium, IN T H E A B S E N C E OR P R E S E N C E OF V E R A P A M I L (250 #g/plate) Compound a
Mutagenicityb in bacterial strain c TA1537 --
ver
TA98 + ver
ver
TAI00 + ver
- ver
+ ver
A nilinoacridines AMSA
16
28"*d
0e
0
0
0
3NH2-AMSA 3NHCOCH3-AMSA 3,6-diNHz-AMSA mAMSA NHCH3-mAMSA 3NHCOOCH3-mAMSA 3CI-mAMSA
0.5 0 0 0.83 0 0 0
5.6* * 0.4** 7.3"* 0.66 0.3"* 0 0
0 0 0 0 0.13 0 0
0 0 0 0 0.12 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0.49 0.09 60 1.4 0
0 0.78* 0.15" 66* 4.0** 0
160 1.3 0 170 0.23 0
240 1.6* 0 240"* 0.56** 0
37 0.9 0 260 0.4 0
40 2.6* 0 350"* 0.8** 0
8.9 5.2 11 2.9 0.7
13"* 4.5 19"* 7.8** 2.6**
0 0 0 0 0
0 0 0 0 0
0 0 0.22 0 0
0 0 0.28* 0 0
Other DNA-binding anticancer drugs Adriamycin Mitoxantrone Ametantrone Nitracrine Bisantrene Quinolinium dibromide (NSC 176 319)
Other acridine derivatives 9-Aminoacridine Quinacrine Proflavine NSC 342 965 NSC 601 316
59 T A B L E 1 (continued) Compound a
Mutagenicity b in bacterial strain c T A 1537
TA98
T A 100
- ver
+ ver
- ver
+ ver
- ver
+ ver
0.3 0
1.5"* 0
0 0
0 0
0 0
0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0.45
0 0 0 0 0 0 0 0 0 0.68**
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
1.1 140
1.7"* 200
38 0
51"* 0
8.9 0
11"* 0
Antiparasitic drugs Ethidium bromide Chloroquine
Tricyclic antidepressants Nortriptyline Doxepen HC1 Amitriptyline Desipramin HCI Protriptyline lmipramine HCI Anatranil Chlorpromazine Tripramine maleate Amoxapine
Biological stains Brilliant cresyl blue Methyl blue Methylene blue Malachite green Nile blue Thymol blue Toluidine blue Rhodamine 123 Pyronine Y Safranin
Hair dyes 4-Nitro-o-phenylenediamine Crystal violet f
Structures are as follows: A M S A , 4"-(9-acridinylamino)methanesulphonanilide; m A M S A , amsacrine, 4"-9(acridinylamino)methanesulphon-m-anisidide; NSC 342 965, N-[2-(dimethylamino)ethyl]acridine-4-carboxamide; NSC 601 316, N-[2-(dimethylamino)ethyl]acridine-4-carboxamide; NSC 176 319, 6-aminoquinolinidum-4-[p-9(4-pyridylamino)phenylcarbamoyl]-anilino-bismethyl dibromide; brilliant cresyl blue, 3-amino-6-diethylamino-2-methylphenoxazinium chloride; crystal violet, hexamethyl-p-rosalin chloride; methyl blue, sodium triphenyl-p-rosaline; methylene blue, tetramethylthionine chloride; nile blue, 3-amino-(1,2-benzo)6-diethylaminophenoxazinium chloride: pyronine Y, tetramethyldiaminoxanthylium chloride; rhodamine 123, N[9-(2-methylcarboxyphenyl)-6-(dimethylamino)-3H-xanthen-3-ylidine]-N-methylmethanaminium chloride; safranin, 3,7-diamino-5-phenylphenazinium chloride; toluidine blue, 3-amino-7-dimethylamino-2-methylphenazathionium chloride. b Mutagenicity is expressed as revertant colonies/tzg c o m p o u n d added to the plate, and is derived from the slope of the corresponding regression equation, ver = verapamil. c Bacterial strains are described in Maron and Ames (1983). d Statistical significance of the differences: **p < 0.001; *p < 0.05. e A value of 0 means that the correlation coefficient of the regression equation is not statistically significant. Strain TA90 (Ferguson et al., 1985) has been used, rather than TA1537, as the former detects weak mutagenicity in this c o m p o u n d while the latter gives negative results (unpublished, this laboratory). a
60 Laboratory using published methods and were pure as judged by thin-layer chromatography. Adriamycin was from Farmitalia Carlo Erba Ltd, Barnet, Herts. (U.K.) and other anticancer drugs were provided by the Park-Davis Division of Warner-Lambert, Ann Arbor, MI (U.S.A.). 9-Aminoacridine and ethidium bromide were from Sigma Chemical Co., St. Louis; proflavine, chloroquine, quinacrine and 4-nitro-ophenylenediamine were purchased from Aldrich Chem. Co., Milwaukee (U.S.A.). The tricyclic antidepressants, originally purchased from the Pharmacy, Auckland Hospital, were purified by Dr. Gerald Woolard, Clinical Chemistry Dept., Auckland Hospital. Pyronin Y and crystal violet were from BDH (U.K.). Other biological stains were obtained from George T. Gurr, London.
Bacterial strains S. typhimurium strains TA98, TA100 and TA1537 were kindly provided by Prof. B.N. Ames (Dept. of Biochemistry, University of California, Berkeley, CA). Upon receipt, all strains were grown to stationary phase and frozen (with 10070 DMSO) in 1-ml aliquots at - 8 0 ° C .
1956). Plates were allowed to harden, and then incubated for 3 days at 37°C before scoring colonies for reversion to histidine independence. A dose range of each compound was tested, and each experimental point performed in triplicate on at least two separate occasions. Reversion characteristics of each strain were tested in each experiment using the disc method of Zieger et al. (1982). The background number of revertants was 8 __ 3 for TA1537, 34 + 7 for TA98, and 171 + 10 for TAI00.
Statistical analysis Mutagenicity data were analysed according to the method of Moore and Felton (1983). A regression line was fitted for the number of mutants versus drug concentration for the linear part of the curve. Mutagenicity was scored as negative if the regression coefficient was non-significant. TO determine whether verapamil had a significant effect on mutagenicity, the slopes ( +__ SD) of the corresponding regression lines with and without verapamil were determined, and the significance of the difference was calculated using Student's t test. Results
Mutagenicity assay For each experiment, a 1-ml vial was removed from the freezer, inoculated into 20 ml fresh medium, and grown for 4 h. Optical density was taken at that time and at quarter hourly intervals thereafter, until a 10-fold dilution into fresh complete medium gave a reading of between 0.11 and 0.12 at 654 nm. (This ensured that all cultures were in the same phase of growth when used.) The S. typhimurium plate-incorporation assay was carried out as described by Maron and Ames (1983). Compounds in no more than 100 /~1 of 50070 ethanol, or 50070 ethanol alone as negative control, were added to 2 ml soft agar containing 5 mM histidine-biotin, maintained at 42°C in a temperature block. 100 ~1 of 2.5 mg/ml verapamil if used, and finally 100 t~l o f the bacterial suspension were added, the tube was mixed and quickly poured over the surface of agar plates containing 20 ml of minimal medium (Vogel and Bonner,
All mutagenicity data, calculated as revertant colonies per /~g of added drug in the presence or absence of verapamil, are summarised in Table 1. Of 43 compounds tested, there were 3 (3NHCOCH3-AMSA, 3NHCH3-mAMSA and 3,6diNH2-AMSA) which were mutagenic only in the presence of verapamil, and one (3NH2-AMSA) whose activity was enhanced more than 10-fold in the presence of verapamil. In each case only frameshift mutagenicity in TA1537 became expressed. 3 N H C H 3 - m A M S A was also weakly mutagenic in TA98 but activity in that strain was not enhanced by verapamil. Several other DNAbinding antitumour drugs were also tested. Adriamycin was mutagenic in TA98 and TAI00 but this was not significantly enhanced by verapamil. In contrast, the mutagenicity of mitoxantrone, ametantrone, nitracrine and bisantrene was enhanced by verapamil. Two acridine an-
61
titumour drugs, NSC 342 965 and NSC 601 316, showed approximately 3-fold enhancement of mutagenicity in the presence of verapamil. Quinolinium dibromide, which binds in the minor groove of DNA (Baguley, 1982) was nonmutagenic. O f the antiparasitic drugs tested, ethidium bromide was mutagenic with a 5-fold enhancement in the presence of verapamil, while chloroquine and quinacrine were non-mutagenic. The mutagenicity of two antibacterial acridine derivatives, 9-aminoacridine and proflavine (3,6-diNH2acridine), was enhanced approximately 2-fold. None of the tricyclic antidepressants tested were mutagenic in any of the bacterial strains used. This was also true for all of the biological stains except safranin, whose mutagenicity in strain TA 1537 was slightly enhanced in the presence of verapamil. The mutagenicity o f one hair dye, crystal violet, was slightly enhanced by verapamil.
Discussion Although none of the compounds we have tested showed any greater enhancement of mutagenicity in the presence of verapamil than the anilinoacridine series originally tested (Baguley and Ferguson, 1986), it is clear that enhancement of mutagenicity is a rather widespread phenomenon, occurring in some representatives of most classes of compounds tested in the present survey. Although we have not carried out DNAbinding studies in all of these compounds, it appears likely that the compounds whose mutagenicity is enhanced must not only be hydrophobic and basic, but must also have a planar polycyclic chromophore with the potential to intercalate between DNA base pairs. Tricyclic antidepressant molecules have a butterfly-shaped chromophore which is generally thought to be incapable of binding strongly to DNA (Palmer et al., 1988). One mutagen which is not a DNA binder is 4-nitro-o-phenylene diamine, which has a different pattern of mutagenicity, and may be reduced to form a chemically reactive compound.
In our original publication (Baguley and Ferguson, 1986), we suggested that verapamil was primarily acting to prevent outward efflux, particularly of basically charged compounds, in both bacterial and eukaryotic cells. Normal epithelial cells may also show the characteristic of verapamilsensitive active efflux of these types of compounds (Thorgeirsson et al., 1987). It is therefore likely that some mammalian cells may show increased sensitivity to carcinogens and environmental mutagens in the presence of verapamil, although it is yet not known whether the tissue concentrations of verapamil in patients on long term treatment are sufficient to increase net uptake. Nevertheless, verapamil should be considered as an agent which could increase risk from some environmental mutagens. To date, we have not tested other calcium channel blockers to determine if they have a similar effect.
Acknowledgements We thank Susan O'Rourke and Pamela Turner for technical assistance. This work was supported bu the Hawke's Bay and Auckland Divisions of the Cancer Society of New Zealand and by the Medical Research Council of New Zealand.
References Atwell, G.J., B.F. Cain, B.C. Baguley, G.J. Finlay and W.A. Denny (1984) Potential antitumor agents, 43. Synthesis and biological activity of dibasic 9-aminoacridine-4-carboxamides, a new class of antitumour agent, J. Med. Chem., 27, 1481-1485. Atwell, G.J., G.W. Rewcastle, B.C. Baguley and W.A. Denny (1987) Potential antitumor agents, 50. In vivo solid tumour activity of derivatives of N[2-(dimethylamino)acridine4-carboxamide, J. Med. Chem., 30, 664-669. Baguley, B.C. (1982) Non-intercalative DNA-binding antitumour compounds, Mol. Cell. Biochem., 43, 167-181. Baguley, B.C., and L.R. Ferguson (1986) Verapamil modulates mutagenicity of antitumour acridines in bacteria and yeast, Biochem. Pharmacol., 35, 4581-4584. Denny, W.A., G.J. Atwell, B.C. Baguley and B.F. Cain (1979) Potential antitumor agents, 29. Quantitative structureactivity relationships for the antileukemic bisquaternary ammonium heterocycles, J. Med. Chem., 22, 134-150. Denny, W.A., B.F. Cain, G.J. Atwell, C. Hansch and A. Pan-
62 thananickal (1982) Potential antitumor agents, 36. Quantitative relationships between experimental antitumor activity, toxicity and structure for the general class of 9-anilinoacridine antitumor agents, J. Med. Chem., 25, 276-315. Ferguson, L.R., W.A. Denny and D.G. MacPhee (1985) Three consistent patterns of response to substituted acridines in a variety of bacterial tester strains used for mutagenicity testing, Mutation Res., 157, 29-37. Hrabowska, M., Z. Mazerska, J. Paradziej-Lukowicz, K. Onoszko and A. Ledochowski (1982) Antitumor activity of l-nitro-9-aminoacridine derivatives, Arzneim. Forsch., 32, 1013-1016. Maron, D., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Moore, D., and J.S. Felton (1983) A microcomputer program for analysing Ames test data, Mutation Res., 119, 95-102. Palmer, B.D., G.W. Rewcastle, G.J. Atwell, B.C. Baguley and W.A. Denny (1988) Potential antitumor agents, 54. Chromophore requirements for in vivo antitumor activity
among the general class of tricyclic carboxamides, J. Med. Chem., in press. Reynolds, J.E.F., and A.B. Prasad (Eds.) (1982) Martindale, The Extra Pharmacopoeia, 28 edn., The Pharmaceutical Press, London. Thorgeirsson, S.S., B.E. Huber, S. Sorrell, A. Fojo, I. Pastan and M.M. Gottesman (1987) Expression of the multidrugresistant gene in hepatocarcinogenesis and regenerating rat liver, Science, 236, 1120-1122. Tsuruo, T., H. Iida, S. Tsukagoshi and Y. Sakurai (1981) Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil, Cancer Res., 41, 1967-1972. Vogel, H.J., and D.M. Bonnet (1956) Acetylornithinase of Escherichia coli: partial purification and some properties, J. Biol. Chem., 218, 97-106. Zeiger, E., D.A. Pagano and I.G.C. Robertson (1981) A rapid and simple scheme for confirmation of Salmonella tester strain phenotype, Environ. Mutagen., 3, 205-209. Communicated by R.J. Preston
57
Elsevier
MTRL 0138
Verapamil as a co-mutagen in the Salmonella/mammalian microsome mutagenicity test L y n n e t t e R. F e r g u s o n a n d Bruce C. B a g u l e y Cancer Research Laboratory, University of Auckland Medical School, Private Bag, Auckland (New Zealand) (Accepted 25 May 1988)
Keywords." Verapamil; Calcium-channel blocking; Anilinoacridines
Summary Verapamil is a calcium-channel blocking agent, commonly used for chronic treatment of heart conditions. We have previously demonstrated that verapamil acts as a co-mutagen in a bacterial mutagenicity test for some experimental anilinoacridine antitumour drugs. Within the anilinoacridines series there are several compounds which are apparently non-mutagenic (or very weak mutagens) in the absence of verapamil, but strong mutagens in its presence. We have now tested a wider range of materials for verapamil enhancement of mutagenicity, to include some of those to which persons on verapamil therapy might be exposed through life-style or occupation. Some verapamil enhancement of mutagenicity was seen with most mutagenic compounds including anticancer drugs, antiparasitic agents, one biological stain and one hair dye. A number of tricyclic antidepressants and biological stains were tested and found to be non-mutagenic. If these results extrapolate to mammalian cells, long-term verapamil therapy could potentially increase the effects of certain enironmental mutagens.
Verapamil is a calcium-channel blocking agent which is used clinically, particularly for the control of various heart complaints such as arrhythmia and angina pectoris (Reynolds and Prasad, 1982). Recommended doses of verapamil may reach 360 mg/day over extended time periods. Verapamil is generally considered to be relatively free from harmful side effects in comparison with other Correspondence: Dr. Lynnette R. Ferguson, Cancer Research Laboratory, University of Auckland Medical School, Private Bag, Auckland (New Zealand).
heart medication (Reynolds and Prasar, 1982). Verapamil is also notable in overcoming resistance to a variety of antibiotics and plant products in multidrug resistant mammalian cells (Tsuruo et al., 1981). In a preliminary study on a group of 3 anilinoacridines and 1 acridine (proflavine), we noted that mutagenicity in the Salmonella mammalian/microsome mutagenicity test could be enhanced, to varying degrees for the different compounds, by verapamil. We also found that the potency of those compounds whose mutagenicity
0165-7992/88/$ 03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
58
was most strongly affected by verapamil in bacteria, was greatly increased by verapamil in a multidrug resistant mammalian cell line (Baguley and Ferguson, 1986). If these effects of verapamil are restricted to a small group of experimental drugs to which the general public are not likely to be exposed, then the phenomenon may be only of theoretical interest. However, if the enhancements are seen with other types of compound in the environment, then they could have rather wider implications for persons on long term verapamil therapy. From our previous studies with anilinoacridine derivatives, it appeared that other strongly basic, hydrophobic molecules would be likely to show similar effects. In this study, we have investigated the ability of verapamil to enhance mutagenicity of
a range of materials, including hair dyes, biological stains, acridine derivatives, tricyclic antidepressants and anticancer drugs. Most of the compounds selected fit the above criteria. Structures of the compounds are described in Table 1, or in either the Merck Index or Reynolds and Prasad, 1982. Materials and methods
Compounds Verapamil was produced by Knoll A.G., Ludwigshafen (Germany). Anilinoacridines (Denny et al., 1982), nitracrine (Hrabowska et al., 1982), NSC 601 316 (Atwell et al., 1987), NSC 176 319 (Denny et al., 1979) and NSC 342 965 (Atwell et al., 1984) were synthesised in the Cancer Research
TABLE 1 M U T A G E N I C I T Y OF VARIOUS C O M P O U N D S IN 3 STRAINS OF S. typhimurium, IN T H E A B S E N C E OR P R E S E N C E OF V E R A P A M I L (250 #g/plate) Compound a
Mutagenicityb in bacterial strain c TA1537 --
ver
TA98 + ver
ver
TAI00 + ver
- ver
+ ver
A nilinoacridines AMSA
16
28"*d
0e
0
0
0
3NH2-AMSA 3NHCOCH3-AMSA 3,6-diNHz-AMSA mAMSA NHCH3-mAMSA 3NHCOOCH3-mAMSA 3CI-mAMSA
0.5 0 0 0.83 0 0 0
5.6* * 0.4** 7.3"* 0.66 0.3"* 0 0
0 0 0 0 0.13 0 0
0 0 0 0 0.12 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0.49 0.09 60 1.4 0
0 0.78* 0.15" 66* 4.0** 0
160 1.3 0 170 0.23 0
240 1.6* 0 240"* 0.56** 0
37 0.9 0 260 0.4 0
40 2.6* 0 350"* 0.8** 0
8.9 5.2 11 2.9 0.7
13"* 4.5 19"* 7.8** 2.6**
0 0 0 0 0
0 0 0 0 0
0 0 0.22 0 0
0 0 0.28* 0 0
Other DNA-binding anticancer drugs Adriamycin Mitoxantrone Ametantrone Nitracrine Bisantrene Quinolinium dibromide (NSC 176 319)
Other acridine derivatives 9-Aminoacridine Quinacrine Proflavine NSC 342 965 NSC 601 316
59 T A B L E 1 (continued) Compound a
Mutagenicity b in bacterial strain c T A 1537
TA98
T A 100
- ver
+ ver
- ver
+ ver
- ver
+ ver
0.3 0
1.5"* 0
0 0
0 0
0 0
0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0.45
0 0 0 0 0 0 0 0 0 0.68**
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
1.1 140
1.7"* 200
38 0
51"* 0
8.9 0
11"* 0
Antiparasitic drugs Ethidium bromide Chloroquine
Tricyclic antidepressants Nortriptyline Doxepen HC1 Amitriptyline Desipramin HCI Protriptyline lmipramine HCI Anatranil Chlorpromazine Tripramine maleate Amoxapine
Biological stains Brilliant cresyl blue Methyl blue Methylene blue Malachite green Nile blue Thymol blue Toluidine blue Rhodamine 123 Pyronine Y Safranin
Hair dyes 4-Nitro-o-phenylenediamine Crystal violet f
Structures are as follows: A M S A , 4"-(9-acridinylamino)methanesulphonanilide; m A M S A , amsacrine, 4"-9(acridinylamino)methanesulphon-m-anisidide; NSC 342 965, N-[2-(dimethylamino)ethyl]acridine-4-carboxamide; NSC 601 316, N-[2-(dimethylamino)ethyl]acridine-4-carboxamide; NSC 176 319, 6-aminoquinolinidum-4-[p-9(4-pyridylamino)phenylcarbamoyl]-anilino-bismethyl dibromide; brilliant cresyl blue, 3-amino-6-diethylamino-2-methylphenoxazinium chloride; crystal violet, hexamethyl-p-rosalin chloride; methyl blue, sodium triphenyl-p-rosaline; methylene blue, tetramethylthionine chloride; nile blue, 3-amino-(1,2-benzo)6-diethylaminophenoxazinium chloride: pyronine Y, tetramethyldiaminoxanthylium chloride; rhodamine 123, N[9-(2-methylcarboxyphenyl)-6-(dimethylamino)-3H-xanthen-3-ylidine]-N-methylmethanaminium chloride; safranin, 3,7-diamino-5-phenylphenazinium chloride; toluidine blue, 3-amino-7-dimethylamino-2-methylphenazathionium chloride. b Mutagenicity is expressed as revertant colonies/tzg c o m p o u n d added to the plate, and is derived from the slope of the corresponding regression equation, ver = verapamil. c Bacterial strains are described in Maron and Ames (1983). d Statistical significance of the differences: **p < 0.001; *p < 0.05. e A value of 0 means that the correlation coefficient of the regression equation is not statistically significant. Strain TA90 (Ferguson et al., 1985) has been used, rather than TA1537, as the former detects weak mutagenicity in this c o m p o u n d while the latter gives negative results (unpublished, this laboratory). a
60 Laboratory using published methods and were pure as judged by thin-layer chromatography. Adriamycin was from Farmitalia Carlo Erba Ltd, Barnet, Herts. (U.K.) and other anticancer drugs were provided by the Park-Davis Division of Warner-Lambert, Ann Arbor, MI (U.S.A.). 9-Aminoacridine and ethidium bromide were from Sigma Chemical Co., St. Louis; proflavine, chloroquine, quinacrine and 4-nitro-ophenylenediamine were purchased from Aldrich Chem. Co., Milwaukee (U.S.A.). The tricyclic antidepressants, originally purchased from the Pharmacy, Auckland Hospital, were purified by Dr. Gerald Woolard, Clinical Chemistry Dept., Auckland Hospital. Pyronin Y and crystal violet were from BDH (U.K.). Other biological stains were obtained from George T. Gurr, London.
Bacterial strains S. typhimurium strains TA98, TA100 and TA1537 were kindly provided by Prof. B.N. Ames (Dept. of Biochemistry, University of California, Berkeley, CA). Upon receipt, all strains were grown to stationary phase and frozen (with 10070 DMSO) in 1-ml aliquots at - 8 0 ° C .
1956). Plates were allowed to harden, and then incubated for 3 days at 37°C before scoring colonies for reversion to histidine independence. A dose range of each compound was tested, and each experimental point performed in triplicate on at least two separate occasions. Reversion characteristics of each strain were tested in each experiment using the disc method of Zieger et al. (1982). The background number of revertants was 8 __ 3 for TA1537, 34 + 7 for TA98, and 171 + 10 for TAI00.
Statistical analysis Mutagenicity data were analysed according to the method of Moore and Felton (1983). A regression line was fitted for the number of mutants versus drug concentration for the linear part of the curve. Mutagenicity was scored as negative if the regression coefficient was non-significant. TO determine whether verapamil had a significant effect on mutagenicity, the slopes ( +__ SD) of the corresponding regression lines with and without verapamil were determined, and the significance of the difference was calculated using Student's t test. Results
Mutagenicity assay For each experiment, a 1-ml vial was removed from the freezer, inoculated into 20 ml fresh medium, and grown for 4 h. Optical density was taken at that time and at quarter hourly intervals thereafter, until a 10-fold dilution into fresh complete medium gave a reading of between 0.11 and 0.12 at 654 nm. (This ensured that all cultures were in the same phase of growth when used.) The S. typhimurium plate-incorporation assay was carried out as described by Maron and Ames (1983). Compounds in no more than 100 /~1 of 50070 ethanol, or 50070 ethanol alone as negative control, were added to 2 ml soft agar containing 5 mM histidine-biotin, maintained at 42°C in a temperature block. 100 ~1 of 2.5 mg/ml verapamil if used, and finally 100 t~l o f the bacterial suspension were added, the tube was mixed and quickly poured over the surface of agar plates containing 20 ml of minimal medium (Vogel and Bonner,
All mutagenicity data, calculated as revertant colonies per /~g of added drug in the presence or absence of verapamil, are summarised in Table 1. Of 43 compounds tested, there were 3 (3NHCOCH3-AMSA, 3NHCH3-mAMSA and 3,6diNH2-AMSA) which were mutagenic only in the presence of verapamil, and one (3NH2-AMSA) whose activity was enhanced more than 10-fold in the presence of verapamil. In each case only frameshift mutagenicity in TA1537 became expressed. 3 N H C H 3 - m A M S A was also weakly mutagenic in TA98 but activity in that strain was not enhanced by verapamil. Several other DNAbinding antitumour drugs were also tested. Adriamycin was mutagenic in TA98 and TAI00 but this was not significantly enhanced by verapamil. In contrast, the mutagenicity of mitoxantrone, ametantrone, nitracrine and bisantrene was enhanced by verapamil. Two acridine an-
61
titumour drugs, NSC 342 965 and NSC 601 316, showed approximately 3-fold enhancement of mutagenicity in the presence of verapamil. Quinolinium dibromide, which binds in the minor groove of DNA (Baguley, 1982) was nonmutagenic. O f the antiparasitic drugs tested, ethidium bromide was mutagenic with a 5-fold enhancement in the presence of verapamil, while chloroquine and quinacrine were non-mutagenic. The mutagenicity of two antibacterial acridine derivatives, 9-aminoacridine and proflavine (3,6-diNH2acridine), was enhanced approximately 2-fold. None of the tricyclic antidepressants tested were mutagenic in any of the bacterial strains used. This was also true for all of the biological stains except safranin, whose mutagenicity in strain TA 1537 was slightly enhanced in the presence of verapamil. The mutagenicity o f one hair dye, crystal violet, was slightly enhanced by verapamil.
Discussion Although none of the compounds we have tested showed any greater enhancement of mutagenicity in the presence of verapamil than the anilinoacridine series originally tested (Baguley and Ferguson, 1986), it is clear that enhancement of mutagenicity is a rather widespread phenomenon, occurring in some representatives of most classes of compounds tested in the present survey. Although we have not carried out DNAbinding studies in all of these compounds, it appears likely that the compounds whose mutagenicity is enhanced must not only be hydrophobic and basic, but must also have a planar polycyclic chromophore with the potential to intercalate between DNA base pairs. Tricyclic antidepressant molecules have a butterfly-shaped chromophore which is generally thought to be incapable of binding strongly to DNA (Palmer et al., 1988). One mutagen which is not a DNA binder is 4-nitro-o-phenylene diamine, which has a different pattern of mutagenicity, and may be reduced to form a chemically reactive compound.
In our original publication (Baguley and Ferguson, 1986), we suggested that verapamil was primarily acting to prevent outward efflux, particularly of basically charged compounds, in both bacterial and eukaryotic cells. Normal epithelial cells may also show the characteristic of verapamilsensitive active efflux of these types of compounds (Thorgeirsson et al., 1987). It is therefore likely that some mammalian cells may show increased sensitivity to carcinogens and environmental mutagens in the presence of verapamil, although it is yet not known whether the tissue concentrations of verapamil in patients on long term treatment are sufficient to increase net uptake. Nevertheless, verapamil should be considered as an agent which could increase risk from some environmental mutagens. To date, we have not tested other calcium channel blockers to determine if they have a similar effect.
Acknowledgements We thank Susan O'Rourke and Pamela Turner for technical assistance. This work was supported bu the Hawke's Bay and Auckland Divisions of the Cancer Society of New Zealand and by the Medical Research Council of New Zealand.
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