Mutagenicity evaluation of the two antimalarial agents chloroquine and mefloquine, using a bacterial fluctuation test

41

Mutation Research, 68 (1979) 41--49 © Elsevier/North-Holland Biomedical Press

MUTAGENICITY EVALUATION OF THE TWO ANTIMALARIAL AGENTS CHLOROQUINE AND MEFLOQUINE, USING A BACTERIAL FLUCTUATION TEST

MARTIN E. SCHUPBACH

Biological and Pharmaceutical Research Department, F. Hoffmann-La Roche and Co., Ltd., Basel (Switzerland) (Received 13 February 1979) (Revision received 3 May 1979) (Accepted 11 May 1979)

Summary The two antimalarial agents chloroquine and mefloquine have been tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1537 and TA1538. Chloroquine was found to revert strain TA1537 at concentrations of 100 and 250 pg/ml, most likely due to intercalation. No mutagenicity was found with mefloquine at concentrations up to 2.5 pg/ml, neither without nor with metabolic activation by Ca2*-precipitated rat liver microsomes. Higher concentrations of mefloquine and chloroquine inactivated the bacteria.

The widely used antimalarial agent chloroquine, which is also used against dermatosis and arthritis, has been reported to inhibit the repair of alkylationand radiation-induced DNA damage as well as normal DNA synthesis in bacterial and mammalian -- including human -- cells [3,7,9,10,14,19,24,25]. Considerable evidence exists which shows that the 3 antimalarial compounds chloroquine, quinine and quinacrine form an intercalated complex with DNA in vitro. On the other hand, mefloquine, another antimalarial agent binds only weakly and does not intercalate at all into calf-thymus DNA in vitro [5]. From these findings a mutagenic effect obtained by shifting the reading frame might be expected for chloroquine. This assumption can best be tested by using different strains of indicator organisms with well~iefined mutations in a reversion assay, as developed e.g. by Ames et al. [1,2]. The same Salmonella Abbreviations: DM, Davls--Mingioli;DMSO, dimethylsulfoxide; G-6-P, D-gtucose 6-phosphate; G-6PDH, glueose-6-phosphate dehydrogenase; NADP, nicotinamide adenine dinueleotide phosphate; NB, nutr/ent broth; NBA, nutrient broth agar.

42 t y p h i m u r i u m strains as in the Ames plating m e t h o d have been successfully used b y Green et al. in a fluctuation test [11,12]. In this paper results are presented which were obtained b y testing chloroquine and mefloquine for mutation induction in different strains of Salmonella t y p h i m u r i u m by a fluctuation test.

Materials and m e t h o d s Bacterial strains Salmonella t y p h i m u r i u m strains TA1535, TA1537 and T A 1 5 3 8 have been

described by Ames et al. [1]. A stock culture containing a low background of his ÷revertants was obtained by the following procedure: 10 single colonies were isolated from streaks on nutrient broth agar (DIFCO) plates, inoculated into 20 ml nutrient broth respectively and incubated overnight in a shaker at 37°C. On the next day 0.1-ml aliquots of each culture were plated on Vogel--Bonner minimal agar + biotin to determine the frequency of his ÷ revertants. Stocks subsequently shown to contain an excessive background of his ÷ revertants were discarded, the other cultures were kept in liquid nitrogen. A 1-ml aliquot was then thawed and grown overnight in 20 ml nutrient b r o t h for each test. Media

NB consisted of DIFCO Nutrient Broth (8 g/l) and NaC1 (4 g/l) in distilled water. NBA was NB plus 15 g/1 agar. DM salts: K2HPO4 (7 g), KH2PO4 (2 g), (NH4)2SO4 (1 g), Na3citrate • 51~ H20 (345 mg), MgSO4 • 7 H20 (100 mg), H20 distilled (1000 ml), autoclaved. For the final DM-medium, the following solutions were sterilized b y filtration and were added to the DM-salt solution immediately before use: 1.25 ml o f a histidine solution (120 pg/ml), 1.25 ml of a biotin solution (6.4 #g/m]), 10 ml o f a glucose solution (400 mg/ml) and 2.5 ml o f a bromocresol solution (2 mg/ml). Solution C: 20 ml MgC12 • 6 H20 (6.1 mg/ml), Tris (Sigma 7--9) (30.3 mg/ ml, pH 7.5), 20 ml sucrose (212 mg/ml), 10 ml H20. The c o m p o n e n t s were autoclaved separately. Animals

Fiillinsdorf albino male rats were fed 0.1% phenobarbital in their drinking water for 5 days before they were killed. The liver of 3 rats was removed and pooled. Preparation o f Ca2÷-precipitated liver microsomes

The procedure o f Frantz and Mailing [8] was followed. The resuspended microsomes were dispensed into 2-ml aliquots and stored in liquid nitrogen for not longer than 4 weeks. The fluctuation test w i t h o u t activation by mammalian liver microsomes

This m e t h o d has been described in detail by Green et al. [11]. 0.25 ml of an overnight culture in NB (ca. 1 × 109 cells per ml), was added to 1000 ml DM-medium. This suspension was divided into several equal portions to which the test com-

43 pound, or the solvent alone, was added (1 ml of the dissolved compound or of the solvent alone to 110 ml medium). Each aliquot was kept on ice and immediately dispensed into 50 small test tubes (2.0 ml per tube equals 5 × 10 s cells per .tube) and incubated at 37 ° C. The auxotrophic bacteria grow until the small supplement of histidine present has been exhausted, thereafter, only his* revertants can continue growth. After 2 days, the tubes in which one or more mutations had occurred, became visibly turbid, while the other tubes remained clear. The addition of the pH indicator bromocresol purple facilitates scoring. The number of positive (yellow) tubes was determined from day 2 to day 6. The readings taken on the 4th day proved to be best for testing the two compounds chloroquine and mefloquine. The fluctuation test with activation by mammalian liver microsomes According to Green et al. [12], the following mixture was prepared: 1 ml DM salts, containing ca. 107 bacteria, 15 pg L-histidine, 0.8 pg biotin and 8 mg glucose, 1 ml solution C, 0.25 ml NADP (5 mg/ml), 0.25 ml G-6-P (8 mg/ml), 1.5 ml DM salts, 0.5 units G-6-PDH, test compound or solvent alone. The mixture was dispensed in 100-pl aliquots into 50 small test tubes per concentration. The tubes were incubated overnight at 37 ° C. On the next day 2 ml of DM medium containing 0.4% glucose and 5 tzg/ml bromocresol purple was added to each tube. Incubation was continued for 3 days. The number of yellow tubes was determined 4 days after the beginning of the experiment. Statistical analysis In the fluctuation test, significance can be tested by X2 [11]. Test compounds Chloroquine diphosphate (7-chloro-4-(4-diethylamino-l-methylbutylamino)quinoline diphosphate) was obtained from Boehringer, Mannheim. Mefloquine hydrochloride (2,8-bis-(trifluoromethyl)~-(2-piperidyl)-4-quinolinemethanol hydrochloride), Lot No. WR 142 490. Benzo[a]pyrene was from Serva. Chloroquine and mefloquine were dissolved in sterile distilled water, benzo [a]pyrene in DMSO (Merck).

" HNCH(CH2) 3 N(C2H5) 2

•~N>"

~ CF3

CF3

I CH 3 Chloroqulne

Mefloqulne

Results

Table 1 summarizes the results of the reversion experiments (fluctuation tests) with chloroquine on 3 different strains of Salmonella typhimurium,

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TABLE 1 SUMMARY OF R E S U L T S OF THE F L U C T U A T I O N TESTS WITH C H L O R O Q U I N E Strain

Significance (Probability)

number

TA1535

0 10 25 50 100 250

1 1 1 1 1 1

4 1 4 1 0 0

TA1537

0

1 2 3

5 2 3

10

1 2 3

4 5 3

25

1 2 3

3 4 7

50

1 2 3

4 5 8

100

1 2 3

12 13 12

P < 0.01 P ~ 0.05

250

1 2 3

50 50 50

P ~ 0.01 P ( 0.01 P ~ 0.01

0

1 2

12 10

10

1 2

10 6

25

1 2

7 8

50

1 2

8 8

100

1 2

4 4

250

1 2

0 0

TA1538

Experiment

Average n u m b e r o f tubes positive ( p e r 50 tubes)

Concentration (~g/ml)

T A 1 5 3 5 , T A 1 5 3 7 and T A 1 5 3 8 , without activation by a mammalian liver microsomal fraction. No mutation induction was observed with strain TA1535 and with strain T A 1 5 3 8 while T A 1 5 3 7 was reverted by chloroquine at concentrations of 100 and 250 pg/ml. The calculation of the accurate mutation rate from a fluctuation test as was used in these experiments, is somewhat problematical [11 ], therefore, such a calculation is omitted here. Mefloquine did not increase the reversion rate of either one of the 3 tester strains T A 1 5 3 5 , TA1537 or T A 1 5 3 8 at concentrations ranging from 0.5 to 2.5 pg/ml. Concentrations above 2.5 #g/ml inactivated the bacteria and could therefore not be tested. No mutation induction was observed, neither without,

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TABLE 2 SUMMARY OF RESULTS OF THE FLUCTUATION SOMAL ACTIVATION

TESTS WITH MEFLOQUINE

Strain

Concentration (/~g/ml)

Experiment number

Average number of tubes positive (per 50 tubes)

TA1535

0

1 2 3

3 4 3

0.125

1 2

4 8

0.250

1 2

2 5

0.500

1 2

6 4

1.250

1 2 3

2 5 2

2.500

1 2 3

5 4 0

0

1 2 3 4

6 1 2 3

0.125

1 2

6 4

0.250

1 2

4 4

0.500

1 2

2 2

1.250

1 2 8 4

6 2 1 4

2.500

1 2 3 4

3 4 4 5

0

1 2 3

4 7 9

0.125

1 2 3

4 2 9

0.250

1 2 3

1 4 9

0.500

1 2 3

3 5 6

1.250

1 2 3

4 3 3

2.500

1 2 3

0 0 4

TA1537

TA1538

WITHOUT MICRO*

Significance (probability)

46 TABLE 3 S U M M A R Y O F R E S U L T S O F T H E F L U C T U A T I O N T E S T S W I T H M E F L O Q U I N E (MQ) W I T H A C T I V I T I O N BY Ca 2+ P R E C I P I T A T E D M I C R O S O M E S A N D P O S I T I V E C O N T R O L W I T H B E N Z O [ a ] P Y R E N E (BP)

Strain

Cornpound

Conceno tration (~Ug/ml)

Experiment number

TA1535

MQ

0

1 2

4 9

0.50

1 2

2 8

1.25

1 2

6 5

2.50

1 2

7 9

0

1 2

13 11

0.50

1 2

4 9

1.25

1 2

17 12

2.50

1 2

11 11

0

1 2

S 4

O.5O

1 2

8 9

1.25

1 2

10 6

2.50

1 2

9 6

0

1 2 3

9 S 6

10

1 2 3

7 11 S

0

1 2 3

4 4 4

10

1 2 3

25 17 24

TA1537

TA1538

TA1538 without microsomes

TA1538 with mierosomes

MQ

MQ

BP

Average n u m b e r o f t u b e s positive (Per 50 t u b e s )

Significance (probability)

P ~ 0.01 P ~ 0.01 P '~ 0 . 0 1

nor after metabolic activation by Ca2+-precipitated rat liver microsomes (Tables 2 and 3). Benzo[a]pyrene served as a positive control for testing the efficiency o f the microsomal preparation. A significant increase over the spontaneous mutation rate was found with this c o m p o u n d at a concentration of 10 pg/ml (Table 3).

47 Discussion

Davidson et al. reported that mefloquine, an antimalarial drug, does not bind to calf-thymus DNA by intercalation [5]. On the other hand, considerable evidence has accumulated showing that chloroquine, which is still quite frequently used as an antimalarial compound, forms an intercalated complex with DNA [4,26]. From these observations one might expect chloroquine to induce frameshift mutations. However, the correlation between intercalating ability and mutagenicity is poor. Many compounds intercalate strongly and have not been found mutagenic [6]. Therefore, intercalation per se might not be sufficient for mutagenesis and it is likely that only particular types of interactions between DNA bases and intercalated compounds can lead to mutations. The results presented in this paper show that intercalation of chloroquine results, in fact, in mutations. Chloroquine reverts strain TA1537 of Salmonella typhimurium. This frameshift mutant has a repetitive G/C sequence at the mutation site and is reverted by mutagens such as 9-aminoacridine and ICR-191. The mutation in strain TA1537 can be suppressed by the frameshift suppressor sufB, which has been shown to be a mutation in the tRNA p~°, suppressing + 1 frameshift mutations of the --CCCC-- type [20]. This is likely to be caused by an added G in the normal --GGG-- anticodon of the proline tRNA, leading to restoration of the proper reading frame. Interaction of chloroquine is apparently much stronger with purine nucleotides than with pyrimidine nucleotides [23]. This fits in with the model proposed by Schreiber and Daune [22]. According to their theory, all frameshift mutations are initiated at G residues to which a dye molecule is bound. This model is attractive because it is consistent with the finding that bacterial frameshift mutations frequently occur in G/C rich sequences. Another frameshift mutant, strain TA1538, is reverted e.g. by various aromatic nitroso derivatives of amines and by 2-nitrosofluorene by a -2 deletion of a -C--G--. Neither this strain nor the base-pair substitution mutant TA1535 could be reverted by chloroquine. When seen as a whole, intercalation appears to be the most plausible explanation for the mutagenic effect of chloroquine, rather than an impaired DNA synthesis. Mefloquine showed no mutagenic activity in neither of the 3 tester strains TA1535, TA1537 or TA1538. The concentrations which could be tested for mefloquine were very low when compared to the minimal concentration of chloroquine required to revert strain TA1537. Concentrations of mefloquine higher than 2.5 pg/ml inactivated the tester bacteria, while with chloroquine the minimal inhibition concentration was above 250 pg/ml. Under the described experimental conditions, there was also no detectable mutagenic metabolite formed by Ca2+-precipitated microsomes. The results of this paper also have a practical aspect. It is well known that mutagens are very likely to be carcinogens [16]. There is a lack of available carcinogenicity data o n chloroquine [13] and to our knowledge no carcinogenicity studies on mefloquine have been published as yet. Because of the high correlation between mutagenicity and carcinogenicity, chloroquine might be expected to induce tumors. Orally administered chloroquine is well absorbed from the gastrointestinal tract in many species and is avidly retained in tissues

48 [ 1 5 , 1 7 ] . The c o m p o u n d is deposited at up to 1 0 0 0 times the plasma concentration in spleen, liver, kidney and lung and it also rapidly crosses the placenta in mice [15,18]. When treatment with chloroquine in man is discontinued, chloroquine and its metabolites can be detected in the urine for up to 5 years [ 2 1 ] . By its action as repair inhibitor in mammalian cells, chloroquine may also increase the effect of other environmental mutagens in individuals taking this drug. It has often been assumed that intercalation into D N A is a prerequisite for the antimalarial activity of a compound. As shown by Davidson et al. [5], the antimalarial agent mefloquine does not intercalate into D N A , probably due to unfavorable steric repulsion caused by the trifluoromethyl groups. Because of the mutagenic and probably also cancerogenic risk o f chloroquine and because of the increasing need for new antimalarial drugs (resistance problem), synthetic chemists should be aware of the fact that other antimalarial c o m p o u n d s exist, as e.g. mefloquine, which do not bind to DNA.

Acknowledgements The author would like to express his thanks to Ms. Esther Kohler, Ms. Yvonne Niinlist, Ms. Annick Wespieser and Mr. Thomas Miiller for their technical help and to Ms. Sheron Bonham and Ms. Ursula Freivogel for assistance with the manuscript. References 1 Ames, B.N., F.D. Lee and W.E. Durston, A n improved bacterialtest system for the detection and classificationof mutagens and carcinogens, Proc. Natl. Acad. Sci. (U.S.A.), 70 (1973) 782--786. 2 Ames, B.N., J. M c C a n n and E. Yamasaki, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-mierosome mutagenicity test,Mutation Res., 31 (1975) 345--364. 3 Cleaver, J.E., and R.B. Painter, Absence of specificity in inhibition of D N A repair replication by DNA-binding agents, cocarcinogens, and steroidsin h u m a n cells,Cancer Res., 35 (1975) 1773--1778. 4 Cohen, S.N., and K.L. Yielding, Spectrophotometric studies of the interaction of chloroquine with deoxyribonucleic acid, J. Biol. Chem., 240 (1965) 3123--3131. 5 Davidson, M.W., B.G. Griggs Jr., D.W. Boykin and W.D. Wilson, Mefloquine, a clinicallyuseful quinolinemethanol antimalarial which does not significantlybind to D N A , Nature (London), 254 (1975) 632--634. 6 Drake, J.W., The molecular basis of mutation, Holden-Day, San Francisco, 1970. 7 Field, R.C., B.R. Gibson, D.J. Holbrook Jr. and B.M. McCall, Inhibition of precursor incorporation into nucleic acids of m a m m a l i a n tissuesb y antimalarialaminoquinolines, Br. J. Pharmacol., 62 (1978) 159--164. 8 Frantz, C.N., and H.V. Mailing, The quantitative microsomal mutagenesis assay method, Mutation Res., 31 (1975) 365--380. 9 Gaudin, D., and K.L. Yielding, Response of a "resistant" plasmocytoma to alkylating agents and X-ray in combination with the "excision repair inhibitors" caffeine and chloroquine, Proc. Soc. Exp. Biol. Med., 131 (1969) 1413--1416. 10 Gaudin, D., K.L. Yielding, A. Stabler and T. Brown, The effect of D N A repair inhibitors on the response of tumors treated with X-ray and alkylating agents, Proc. Soc. Exp. Biol. IVied.,137 (1971) 202--206. 11 Green, M.H.L., W.J. Muriel and B.A. Bridges, Use of a simplified fluctuation test to detect low levels of mutagcns, Mutation Res., 38 (1976) 33--42. 12 Green, M.H.L., B.A. Bridges, A.M. Rogers, G. Horspool, W.J. Muriel, J.W. Bridges and J.R. Fry, Mutagen screening b y a simplified bacterial fluctuation test: Use of microsomal preparations and whole liver cellsfor metabolic activation,Mutation Res., 48 (1977) 287--294. 13 I A R C Monograph on the evaluation of carcinogenic risk of chemicals to man, Vol. 13, Int. Agency Res. Cancer, Lyon, 1977. 14 Kim, S.H., J.H. K i m and J. Fried, Enhancement of the radiation response of cultured tumor cellsby

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chloroquine, Cancer, 32 (1973) 556--540. 15 Lindquist, N.G., A c c u m u l a t i o n of drugs on melanin, Acta Radiol., Suppl. 325 (1973) 1--92. 16 McCann, J., E. Choi, E. Yamasaki and B.N. Ames, Detection of carcinogens as mutagens in the SalmoneUa/microsome test: Assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72 (1975) 5135-5139. 17 McChesney, E.W., and J.P. McAuliff, Laboratory studies of the 4-aminoquinollne antimalarials, I. Some b iochemical characteristics of chloroquine, h y d r o x y c h l o r o q u i n e and SN-7718, A nt i bi ot . Chemother., 11 (1961) 800--810. 18 McChesney, E.W., W.F. Banks Jr. and R.J. Fabian, Tissue d i s t r i b u t i o n of chloroquine, h y d r o x y chloroquine and deset hylchloroquine in the rat, Toxicol. APpl. Pharmacol., 10 (1967) 501--513. 19 Michael, R.O., and G.M. Williams, Chloroquine i n h i b i t i o n of repair of DNA damage induced in m a m m a l i a n cells b y m e t h y l m e t h a n e s u l f o n a t e , Mutation Res., 25 (1974) 391--396. 20 Riddle, D.L., and J.R. Roth, Frameshift suppressors, III. Effects of suppressor m u t a t i o n s on transfer RNA, J. Mol. Biol., 66 (1972) 495--506. 21 Rubin, M., H.N. Bernstein and N.J. Zvaifler, Studies on the pharmacology of chloroqulne: Recomm e n d a t i o n s for the t r e a t m e n t of chioroquine r e t i n o p a t h y , Arch. Ophthalmol., 70 (1963) 474--481. 22 Schreiber, J.P., and M.P. Daune, Fluorescence of complexes of acridine dye w i t h synthetic polydeo x y r i b o n u c l e o t l d e s : A physical model of framashift mutagenesis, J. Mol. Biol., 83 (1974) 487--501. 23 Sternglanz, H., K.L. Yielding and K.M. Pruitt, Nuclear magnetic resonance studies of the i n t e r a c t i o n of chloroquine diphosphate with adenosine 5'-phosphate and other nucleotides, Mol. Pharmacol., 5 (1969) 376--381. 24 Whichard, L.P., M.E. Washington and D.J. Holbrook Jr., The i n h i b i t i o n in vitro of bacterial DNA polymerases and RNA Polymerase by antimalarial 8-aminoquinoUnes and b y chloroquine, Biochim. Biopbys. Acta, 287 (1972) 52---67. 25 Yielding, K.L., L. Yielding and D. Gaudin, I n h i b i t i o n b y chloroquine of UV repair in E. coli B', Proc. Soc. Exp. Biol. Med., 133 (1970) 999--1001. 26 Waring, M., Variation of the supercoils in closed circular DNA b y bi ndi ng of antibiotics and drugs: Evidence for molecular models involving intercalation, J. Mol. Biol., 54 (1970) 247--279.