Aspects of chloroquine mutagenicity

Mutation Research, 175 (1986) 51-59 Elsevier

51

MRLett. 0898

Aspects of chloroquine mutagenicity E.E. Obaseiki-Ebor* and E.E. Obasi Department of PharmaceuticalMicrobiology, Faculty of Pharmacy, Universityof Benin, Benin City (Nigeria)

(Accepted 16 May 1986)

Summary Using the Ames plate reversion and fluctuation tests, the mutagenic activity of chloroquine was tested in the new tester strains of Salmonella typhimurium, TA97, TA102, and Escherichia coli strains WP2, WP2hcr, WP6 and WP67. The E. coli transconjugants obtained from the mating transfer of R-plasmid(s) in strains TA97 and TA102 respectively to E. coli WP2, i.e. EE97 and EEl02, were also tested. Chloroquine reverted strain TA97 from histidine dependence to independence and also reverted E. coli strains EE97 and EEl02 from tryptophan dependence to independence. The E. coli strains WP2, WP2hcr; WP6 and WP67 and S. typhimurium TA102 were not affected. S. typhimurium TA97 could be reverted with 250 ng/ml of chloroquine (therapeutic blood level of chloroquine is 300 ng/ml). Reversion generally occurred optimally at the relatively lower concentrations of chloroquine i.e. 25, 50/~g/ml than at higher concentrations. From the properties of the reverted tester strains, the results indicated that chloroquine per se mediated frameshift reversion.

Chloroquine is commonly used therapeutically in Nigeria and other countries where malaria is endemic. Chloroquine is deposited in high concentrations in mammalian tissues, it can cross the placenta (Lindquist, 1973; McChesney et al., 1961, 1967) and the metabolites may remain in the body for up to 5 years (Rubina et al., 1963). Chloroquine is known to form intercalated complexes with DNA (Cohen and Yielding, 1965; Steuglanz et al., 1969; Waring, 1970), act as an inhibitor of both normal DNA synthesis and repair of bacterial and human cells (Field et al., 1978; Yielding et al., 1970; Wichard et al., 1972; Michael and Williams, 1974). * Correspondence.

Reported results of chloroquine mutagenicity tests have been contradictory. While Schupbach (1979) reported that chloroquine exhibited frameshift histidine reversion mutation in Salmonella typhimurium TA1537 using the Ames fluctuation test, Middleton and Wiseman (1981), using the agar-plate reversion tests, reported that chloroquine was not mutagenic in Salmonella typhimurium TA1537, TA1536 and TA90. In this paper, chloroquine mutagenicity was determined based on the Ames fluctuation and plate-reversion tests (Ames et al., 1973, 1975). The bacterial tester strains used were the internationally recognised newer and improved Salmonella typhimurium TA97 and TAI02 strains (Maron and Ames, 1983), the E. coli WP2; WP2hcr, WP~ and WP67 strains

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52 and the E. coli transconjugants obtained (in this laboratory) from the transfer of R-plasmids from the Salmonella typhimurium strains TA97 and TA102, respectively, to E. coli WP2. Materials and methods

Bacterial strains The Salmonella typhimurium his- strains used were TA97 and TA102. TA97 contains the Rplasmid pkM101 (which enhances error-prone DNA repair and confers ampicillin resistance); it has been proposed as a replacement for TA1537 (Levin et al., 1982; Maron and Ames, 1983) and it detects frameshift mutagens that act at rich G.'C sequences. In addition to R-plasmid pkM101, TA102 contains another multicopy R-plasmid pAQ 1 which carries the his G428 mutation and a tetracycline resistance gene. TA102 detects mutagens that require an intact excision-repair system and oxidative mutagens that preferentially act at A:T base pairs. The E. coli strains were WP2 trp, WP2 uvrA; WP6 trp, polA1; WP67 trp, polA1; uvrA. These strains detect base-pair substitution mutagens at the trp mutant site. The E. coli strains EE97 and EEl02 were transconjugants obtained from the conjugation transfer of R-plasmids from Salmonella typhimurium strains TA97 and TA102, respectively, to the E. coli wild-type WP2. Transconjugants were selected on McConkey agar containing 50 #g ampicillin/ml. The transconjugants were purified and tested for their dependence on histidine or tryptophan for growth in minimal medium and their resistance to ampicillin alone and, ampicillin plus tetracycline, respectively. Single isolated colonies of all the tester strains were subcultured onto nutrient agar slants and stored at 4°C. Media Nutrient broth No. 2 (Oxoid), nutrient agar (Oxoid), agar No. 1 (Oxoid), and Davis and Mingioli salts solution (1951) were used. The Davis and Mingioli salts solution used for all the mutagenici-

ty tests generally contained 62.5 ng/ml tryptophan or histidine in trace amounts except as otherwise stated. This low concentration of tryptophan or histidine would stimulate the growth of the auxotrophic cells until it is exhausted; thereafter, only trp + or his + revertants could continue to grow.

Drug Chloroquine diphosphate (Sigma Chem. Co.) was dissolved in distilled water at a concentration of 10 mg/ml. It was sterilised by autoclaving at 115°C for 30 min at 10 lb./square in. Acridine orange (Sigma Chem. Co.) and nitrosoguanidine (Sigma Chem. Co.) were used as control test substances. Test culture The bacteria tester strains were grown overnight in 10 ml nutrient broth to a culture density of about 2 x l0 s cells/ml. The cultures were harvested and washed 3 times with sterile distilled water and resuspended in 10 ml sterile distilled water. Minimum inhibitory concentration determination (MIC) The minimum inhibitory concentration of chloroquine against the test bacteria strains was determined by the agar-dilution protocol previously described (Obaseiki-Ebor, 1984). Wet-dried Davis and Mingioli minimal agar (supplemented with tryptophan or histidine) plates containing various concentrations of chloroquine ranging from 10 #g/ml to 1 mg/ml were inoculated with about 105-106 cfu of the washed test cultures. The MIC was the lowest concentration of chloroquine that completely inhibited growth after 24 h incubation at 37°C. Mutagenicity test determinations (a) Spot test. A modified method of Brusick et al. (1980) was used. 106 cfu/ml of the test culture were spread on the Davis and Mingioli minimal agar plates (unsupplemented), and a few crystals (10 mg) of chloroquine were placed on the surface of the agar. A few

53

crystals (10 mg each) of nitrosoguanidine and acridine orange were placed on similar inoculated plates as positive controls. The plates were incubated at 37°C for 48 h and scored for the number of revertant colonies and/or any bacteria inhibitory zone which might be present. The means of triplicate results were recorded. (b) Plate-incorporation test. A modified agar overlay test method as previously described (Brusick et al., 1980) was used. Various subinhibitory concentrations of chloroquine (25, 50, 100, 200 ug/ml) were added to tubes containing 2.5 ml of molten overlay agar (0.7070 agar) held at 45°C. 0.1 ml of a 10-5 dilution of the test culture were added to the tubes, the contents of the tubes gently mixed, poured onto the surface of Davis and Mingioli agar plates and allowed to solidify. Controls, positive (containing tryptophan 50 #g/ml or histidine 50 #g/ml and no test agent) and negative (not containing amino acids and test agent) were included. All plates were incubated at 37°C for 48 h and the number of colonies growing per plate in mean triplicate results were recorded.

Modified fluctuation test The method of Green et al. (1976) was suitably modified. A fresh overnight washed test culture in 10 ml Davis and Mingioli liquid medium appropriately supplemented with either histidine (50 ttg/ml) or tryptophan (50/~g/ml) and glucose (200 /~g/ml) was grown to log phase, harvested and washed free of the histidine or tryptophan supplements. 0.4 ml of the resuspended culture (1 x 109 cfu/ml) in 10 ml sterile distilled water was inoculated into 400 ml Davis and Mingioli basal salts containing glucose (200 #g/ml), biotin (5 #g/ml) and bromocresol purple indicator (1.2×10 -2 mg/ml). The contents were thoroughly mixed and aseptically divided into 4 portions of 100 ml as follows: 100-ml aliquot containing test concentration of chloroquine (in duplicate), 100-ml aliquot containing additional concentration of histidine or tryptophan (50/zg/ml), 100-ml aliquot containing no histidine, tryptophan and test chloroquine. The contents of each flask were aseptically

dispersed in 2-ml aliquots into 50 small test tubes and incubated at 37°C. After 72 h of incubation, the tubes containing his ÷ or trp + revertants were turbid, which meant their colour changed from purple to yellow. Each duplicate test concentration of chloroquine (25, 50, 100 and 200 ~g/ml) was determined on at least two occasions.

Statistical analysis The results from the fluctuation tests were evaluated for significance using the following X2 (chi-square) test. 2N (t - c)2

X2 =

(t + c) 2n - t - c where N = number of tubes in the control and treated set, t = number of revertant tubes (yellow) in the treated set, c = number of revertant tubes (yellow) in the control set. Based on 50 tubes per set, with 1 degree of freedom, significance at 5070 level requires: X2 = 3.84 and at the 1070 level x2>6.63. Results

The minimum inhibitory concentration of chloroquine against the test Salmonella typhimurium and the E. coli strains was in excess of 1 mg/ml in the appropriately supplemented Davis and Mingioli minimal agar plates. There was, however, a remarkable inhibition of growth at concentrations beyond 250 ~g/ml compared to the good growth in the plates containing less than 250 /~g/ml of chloroquine. Thus, the subinhibitory concentrations of 25, 50, 100 and 200 #g/ml of chloroquine were used as the test concentrations in this study. The spot test experiments (Table 1) showed that chloroquine could induce a clear zone of inhibition and revert the growth of S. typhimurium TA97 from histidine dependence to independence (histo his+), similar to the activities of acridine orange and nitrosoguanidine against S. typhimurium TA97 and the E. coli strains (trp- to trp+), respectively. However, S. typhimurium TA102 was not

54 TABLE 1 SUMMARY OF RESULTS OF THE SPOT-TEST WITH CHLOROQUINE COMPARED TO ACRIDINE ORANGE AND NITROSOGUANIDINE Mutagen

Strains ( x I0 e cfu)

Number of revertant colonies a

Diameter a of zone of

Control plate

Test plate

inhibition (ram)

Chloroquine

TA97 TA102 WP2 WP2hcr WP6 WP67

4 3 7 3 3 5

111 4 10 5 4 6

43 NR NR NR NR NR

Acridine orange

TA97 TA102 WP2 WP2hcr WP6 WP67

2 4 10 5 2 3

150 3 9 6 4 3

48 NR NR NR NR NR

Nitrosoguanidine

TA97 TAI02 WP2 WP2hcr WP6 WP67

4 2 12 9 3 6

3 4 128 73 35 55

NR NR 45 41 55 38

a Values are mean of triplicate experiments. NR, no ring or inhibitions were obtained.

reverted by chloroquine, acridine orange or nitrosoguanidine. In the plate incorporation reversion test experiments (Table 2a), chloroquine reverted S. typhimurium TA97 without reverting TA102 strains. E. coli WP2 strains were also not reverted from trp- to trp + but the E. coli WP2 transconjugant strains EE97 and EEl02 (obtained from the transfer of the R-plasmids in S. typhimurium

strain TA97 and TA102 to WP2, respectively) were reverted from trp- to trp +. Generally, the number of revertants decreased as the concentration of the test chloroquine increased with optimum reversion in the range 25-50 t~g/ml of chloroquine. Lower concentrations of chloroquine at ng/ml concentrations were found to revert strain TA97 i.e. at 250 ng/ml (Table 2b).

TABLE 2a PLATE-INCORPORATION TEST RESULTS WITH CHLOROQUINE Strain

Medium

Concentration ~g/ml)

Number of revertant colonies per plate

TA97

histidine (50 ttg/ml) histidine (absent)

0 0 25 50 100 200

282 0 172 133 73 23

55

TABLE 2a (continued) Strain

Medium

Concentration ~g/ml)

Number of revertant colonies per plate

TAI02

histidine (50 ttg/ml) histidine (absent)

0 0 25 50 100 200

294 0 2 5 7 4

WP2

tryptophan (50/~g/ml) tryptophan (absent)

0 0 25 50 I00 200

274 0 1 0 0 0

WP2hcr

tryptophan (50 ~tg/ml) tryptophan (absent)

0 0 25 50 100 200

296 2 8 6 4

3

WP6

tryptophan (50/tg/ml) tryptophan (absent)

0 0 25 50 lO0 200

300 0 1 l 2 6

WP67

tryptophan (50/tg/ml) tryptophan (absent)

0 0 25 50 I00 200

288 2 4 3 3 3

EE97

tryptophan (50 ~,g/ml) tryptophan (absent)

0 0 25 50 I00 200

228 0 II 4 2 I

EEl02

tryptophan (50 ~g/ml) tryptophan (absent)

0 0 25 50 100 200

179 0 1 4 2 1

56 TABLE 2b PLATE-INCORPORATION TEST RESULTS WITH CHLOROQUINE AT LOWER (ng/ml) CONCENTRATIONS Strain

Medium

Concentration of chloroquine

Number of revertant colonies per plate

TA97

histidine (50/~g/ml) histidine (absent)

0 0 10 ng/ml 50 100 250 500 1 /zg/ml 5 15

280 0 0 0 2 8 8 10 12 16

EE97

tryptophan (50/~g/ml) tryptophan (absent)

0 0 10 ng/ml 50 100 250 500 ! #g/ml 5 15

288 0 1 1 1 0 0 0 1 2

EEl02

tryptophan (50 t~g/ml) tryptophan (absent)

0 0 10 ng/ml 50 100 250 500 1 /~g/ml 5 15

274 2 3 3 1 3 1 2 1 1

In the fluctuation test, chloroquine also reverted strain TA97 without affecting strain TA102 (Table 3), while the E. coli strains EE97 and EEl02 were reverted without the reversion of WP2. The fluctuation test reversion in TA97 and EEl02 decreased as the concentration of chloroquine increased, while in EE97, reversion increased as the concentration of chloroquine increased. Discussion

Antimalarial drugs are frequently administered

to humans in tropical regions of the world. This raises the pertinent desirability that this group of drugs should be free from possible mutagenic or carcinogenic effects. The mutagenic activity of chloroquine has not been conclusively shown since varying results have been obtained with various tester strains (Schupbach, 1979; Middleton and Wiseman, 1981). In this study, using some of the recently internationally recommended bacterial mutagenicity tester strains (Maron and Ames, 1983), chloroquine was found to mediate frameshift mutation. In this study, using the spot-test

57 TABLE 3 FLUCTUATION TEST RESULTS WITH CHLOROQUINE AGAINST Salmonella typhimurium STRAINS TA97 AND TAI02 Strain

Salmonella typhimurium TA97

Salmonella typhimurium TA102

Chloroquine Average number of tubes positive in 50 tubes containing concentration Ozg/ml) Chloroquine (present), Chloroquine (absent), Chloroquine (absent), Significance histidine (absent) histidine (present) histidine (absent) (probability) 0

0

50

0

25 50 100 200

35 33 23 15

50 50 50 50

1 0 0 1

0 25 50 100 200

0 0 1 0 0

50 50 50 50 50

0 0 1 1 0

P < 0.01 P < 0.01 P<0.01 P<0.01

TABLE 4 FLUCTUATION TEST RESULTS WITH CHLOROQUINE AGAINST E. coli WP2 AND THE R-PLASM1D FROM S. typhimurium TA97 AND TAI02 TRANSCONJUGANTS Strain

E. coil WP2

Chloroquine concentration ~g/ml)

Average number of tubes positive in 50 tubes containing Chloroquine (present), Chloroquine (absent), Chloroquine (absent), Significance tryptophan (absent) tryptophan (present) tryptophan (absent) (probability)

0 25 50 100 200

2 1 0 0 1

50 50 50 50 50

1 0 0 0 0

P<0.01

E. coli WP2 (pkMl01) (EE97)

0 25 50 100 200

2 4 6 6 7

50 50 50 50 50

2 2 1 0 2

P<0.01 P < 0.01 P < 0.01 P<0.01 P<0.01

E. coli WP2 (pkM101) (EEl02)

0 25 50 100 200

4 14 18 13 14

50 50 50 50 50

0 1 2 0 2

P<0.01 P<0.01 P<0.01 P<0.01 P < 0.01

method, chloroquine compared favourably with the spot-test frameshift reversion results of S. typhimurium TA97 strain by acridine orange and contrasted with the results of nitrosoguanidine against E. coli WP2 strains (base-pair substitution mutants).

P<0.01 P<0.01

If the tester strains were confined to only E. coli WP2 strains and to the now obsolete S. typhimurium TA1537, TA1536 and TA90 strains using the agar-plate reversion tests (Middleton and Wiseman, 1981), the results of this study may well have been different. S. typhimurium TA97 has

58 replaced the less sensitive frameshift mutant TA1537 (Levin et al., 1982; Maron and Ames, 1983). In a preliminary experiment in this study, chloroquine mutagenesis was not observed in strain TA1537 confirming the earlier results of Middleton and Wiseman (1981). Chloroquine reverted strain TA97 of S. typhimurium from histidine dependence to independence by all methods employed in the study. S. typhimurium strain TA102 was not reverted by chloroquine although, in addition to the plasmid pkM101, it contained another plasmid pAQI, both of which detected frameshift mutation. E. coli WP2 strains (which detect base-pair substitution mutagens) were not mutagenised by chloroquine, but on the respective transfer of plasmids pkM101 (in TA97), and pkM101 and pAQ1 (in TA102) into E. coli WP2, chloroquine mutagenicity was observed. The inability of chloroquine to mutagenise strain TA102 (a frameshift mutant) could be related to the mechanism of mutagenic activity by chloroquine, since strain TA102 detects mutagens that require an intact excision repair system. The effect of the pkM101 and pAQ1 plasmids in enhancing the mutagenic activity of E. coli WP2 could be in line with the postulation by McCann et al. (1975) that some mutagenic plasmids may have cryptic properties which, on transfer, cause a loss of the lipopolysaccharide barrier that coats the surface of the host bacteria cell. Although the mechanism of chloroquine-induced mutagenesis is not yet understood, the increases and decreases in mutagenic response of the tester strains to different concentration ranges of chloroquine in this study were of interest, and could be considered to be related to the concentration of chloroquine effecting optimum mutagenesis and the effect of the excess chloroquine (more than the concentration required to elicit optimum mutagenesis) on the reversion response and cell survival of the tester strains. Optimum chloroquine-induced frameshift reversions in strains TA97 and EEl02 were within the range 25-50 ttg/ml irrespective of the test method, with higher concentrations eliciting a decreasing response, probably due to chloroquine toxicity on the reverse mutation processes leading

to the prototrophy of the tester strains. However, in strain EE97, increasing reversions were obtained with increasing concentrations of chloroquine due to the cryptic effects of plasmid pkMl01 which could have been enhancing the chloroquine permeability of strain EE97 with further amplification of the mutagenic response. It seems that chloroquine permeability of E. coli is more efficient than that of Salmonella typhimurium. Hence, the observed difference in the responses of EEl02 and TA102 to chloroquine mutagenicity since they both contain the same plasmids. Further studies on the chloroquine uptake by these tester strains will elucidate these postulations. The mutagenic effect of chloroquine also indicates that an initial stimulation of the replication of the target DNA cell is required. Preliminary studies indicated that a trace amount of histidine or tryptophan was always required in the medium before mutagenic activity was observed. The trace amount of about 67 ng/ml of tryptophan or histidine was found to be adequate. This concentration could stimulate growth of the cells, thereby enhancing the intercalation of chloroquine into the DNA base and induce mutation (reversion from his- to his ~ or trp- to trp ÷) while the unmutated cells discontinue growth on exhaustion of the trace chemicals. The therapeutic blood level of chloroquine is variably about 150-250 ng/ml (Goodman and Gillman, 1975), although in malarial patients undergoing treatment with chloroquine, concentrations much higher than 300 ng/ml could be obtained in the blood (Adelusi et al., 1982), and from this study 25-50 #g/ml chloroquine was found to revert strain TA97 indicating that frameshift mutation could be induced by chloroquine at relatively low therapeutic concentrations. Further work on the mutagenic activity of chloroquine metabolites is expected to resolve the risk of chronic chloroquine administration (Lindquist, 1973; McChesney and McAuliff, 1961), since chloroquine is mutagenic in vitro and accumulates in the body tissues. On the contrary, preliminary tests have shown that the chloroquine metabolites (desethylchloroquine and bisdesethylchloroquine) were not

59

mutagenic to these bacterial tester strains. This, therefore, indicates that although chloroquine per se could be mutagenic, the chloroquine metabolites have so far not shown any mutagenic activity.

Acknowledgement This work was supported by the University of Benin Staff Research Grant C/60.

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