241

Mutation Research, 77 (1980) 241--244 © Elsevier/North-Holland Biomedical Press

MUTAGENICITY OF NIFURTIMOX IN Escbericbia coli

T. OHNISHI, Y. OHASHI, K. NOZU and a S. INOKI

Department of Biology and a Department of Pa:asitology, Nara Medical University, Kashihara, Nara 634 (Japan)

(Received 17 July 1979) (Revision received 15 October 1979) (Accepted 17 October 1979)

Summary

The effects of nifurt imox, a nitrofuran derivative, on killing and mutat ion induction in 3 Escherichia coli strains having different DNA-repair systems for UV lesion were studied and compared with the effects o f furylfuramide. Nifur- t imox induces mutat ions at a high frequency in both Hs30R (uvrA-) and H/ r30R (radiation-resistant), although no significant killing effect is detected with Hs30R. No significantly induced mutat ion frequency could be detected with NG30 (recA-), which is very sensitive to killing by nifurt lmox. The characteristics of lesions of DNA induced by nifurt imox and the mechanism of mutat ion induction in Hs30R are discussed.

It is known that some nitrofurans are mutagenic in bacteria [5,6,10] and in silkworms [9], and that they induce cancers in animals [1] and chromosomal aberrations in human lymphocytes [11]. However, nifurt imox (3-methyl-4-(5'- nitrofurylidene-amino)-tetrahydro-4H-1,4-thiazine-1,1-dioxide (Lampit®)), which is active against bo th acute and chronic Chagas' infections in animals and man [2], has not ye t been reported as mutagenic in bacteria. We have examined whether nifurt imox induces back mutat ions (arg-~ arg ÷) in wild-type Esche- richia coli (radiation-resistant) and in a radiation-sensitive mutant (uvrA-), using for comparison a well-known mutagen, furylfuramide [3]. Comparative studies on the cell-inactivating properties of the two compounds were also performed.

The E. coli strains used were H/r30R (argF~mber) , Hs30R (argFarnber , uvrA-) and NG30 (argF~mber, recA-). They were obtained from Dr. S. Kondo, Osaka University. For bacterial growth, a broth medium (10 g polypeptone , 10 g meat extract and 2 g NaC1 in 1 1 of water, pH 7.4) was used. Davis minimal plates (7 g K2HPO4, 2 g KH2PO4, 0.1 g MgSO4, 1 g (NH4)2SO4, 0.5 g sodium citrate

101

and 20 g agar in 1 1 of water), supplemented with a small amount of glucose and Difco nutr ient broth at a final concentration of 0.2 and 0.008% resp., were prepared and used for the mutat ion assays.

Nifurtimox was supplied by Bayer AG, Leverkusen (Germany). Nifurtimox and furylfuramide were dissolved in dimethylsulfoxide (DMSO) at a concentra- tion of 5 mg/ml and diluted with 0.9% saline before use.

E. coli cells in growth phase were used. Saline (0.9%) was used for washing and resuspending. In killing tests, cells were suspended in saline after being washed twice at a concentration of 6 X 108 cells/ml. An equal volume of the drug solution was mixed with the cell suspension at various concentrations. After t reatment for 1 h at 37°C, the mixture was spread on broth plates containing 2% agar, and the number of colonies was counted after 24 h at 37°C. In mutat ion tests, after incubation of the bacteria for 1 h with the drug, 0.1-ml aliquots were spread on Davis minimal plates with 2 ml of supplemented Davis minimal medium containing 0.3% agar. The number of revertants was counted after 48 h at 37 ° C.

The results of killing tests are shown in Fig. 1. The radiation-sensitive strain NG30 was most sensitive to killing by nifurt imox and also by furylfuramide, but the other radiation-sensitive strain Hs30R was almost as resistant to nifur- t imox as the radiation-resistant strain H/r30R, although Hs30R was very sensitive to furylfuramide.

The results of mutat ion tests are shown in Table 1. Nifurtimox induced back mutat ions to arginine independence at a high frequency in two strains, Hs30R and H/r30R. The frequency of induced arg* mutants went up with increasing

NIFURTIMOX

N

;6a l 0

242

I I 100 200

CONCENTRATION OF

FURYLFURAMIDE

I I 0 0.1 0.2

CHEMICALS ( ,pg/rnl )

Fig. 1. Survival curves o f 3 der ivat ives o f E. coli af ter t r e a t m e n t w i t h n i f u r t i m o x and f u r y l f u r a m i d e . T h e f inal c o n c e n t r a t i o n o f the d r u g in t h e r e a c t i o n m i x t u r e is i n d i c a t e d o n t h e abscissa, o I H / r 3 0 R ; ~, H s 3 0 R ; o, N G 3 0 .

243

T A B L E 1

R E S U L T S O F M U T A T I O N TES TS W I T H T H E C H E M I C A L S N I F U R T I M O X A N D F U R Y L F U R A M I D E IN 3 S T R A I N S OF E. coli

Strain Concentrat ion Survival N u m b e r o f I n d u c e d mutat ion o f chemica l pe r ml r eve r t an t s frequency (~g /ml ) ( × 1 0 -6) pe r 0.1 ml (XIO 8)

N i f u r t i m o x H / r 3 0 R 0 353 2 5.7

50 339 13 38.3 100 263.5 11 41.7 250 118 12 101.7

Hs30R 0 323 .5 3 9.3 50 253 43 170 .0

100 148 70 473 .0 250 139.5 81 580.6

N G 3 0 0 396 1.5 3.8 10 367 1 2.7 20 312 0 .5 1.6 30 148.5 0 0 40 99 0 0 50 52 0 0

F u r y l f u r a m i d e

H / r 3 0 R 0 247 2 8.1 0 .05 234 3.5 15 .0 0.1 176 10.5 60 .0 0.2 104 7 .5 72.1 0.3 37.2 5 134 .4

H s 3 0 R 0 220 2.5 11 .4

0 .025 158 146 .5 9 2 7 .2 0 .05 53 156 2 943 .4 0.1 5 32 .5 6 500.0 0 .2 0 .58 11 18 965 .5

N G 3 0 0 156 .5 1.5 9.6 0 . 0 2 5 105 1 9.5 0 .05 62 0 0 0.1 12.5 0 0 0.2 0 .25 0 0

Each va lue r ep resen t s the average o f dupl ica te e x p e r i m e n t s .

dose of nifurtimox. In strain Hs30R, the frequency of revertants increased more than 60 times over the control at concentrations of 250/ag nifurt imox per ml; this frequency was about 5 times higher than that induced at the same nifurt imox concentration in strain H/r30R. No significant increase in mutat ion frequency was detected in strain NG30. Analogous results were obtained with furylfuramide, but the induced mutat ion frequencies were higher when this compound was used. There was also a large difference (260 times or more at 0.2 ~g/ml) in mutat ion frequency between the two strains, H/ r30R and Hs30R.

In the present experiments, 3 strains of E. coli were used having different repair mechanisms for ultraviolet-induced lesions in DNA. It is well known that many kinds of chemical as well as UV irradiation induce mutat ions in micro- organisms [8,12]. In radiation-resistant strains, the UV lesion (pyrimidine dimers) is efficiently removed from the DNA and the gaps resulting from the

244

removal are correctly repaired through several successive enzymic steps [7] . Ix radiation-resistant cells, essentially the same mechanism can operate on the chemical lesions induced in DNA by furylfuramide [3] or 4-nitroquinoline-1 oxide [4] . The present results, however, show that the uvrA derivative, Hs30R is rather resistant to the killing effects of nifurtimox, indicating that the inac. tivating lesions induced by nifurtimox are not recognized by the uvrA gene product. On the other hand, it is clear that the recA gene product is required for mutation induction with nifurtimox, indicating that daughter-strand gap. filling [13] may be the mutation inductibn process responsible. But there is les~, difference in mutation frequency with nifurtimox between strains H/r30R and Hs30R (about 5 times) than with furylfuramide. The presence of uvrA gene product, even in the presence of recA gene product, seems to have little effect on the mutagenic response due to nifurtimox treatment of strains H/r30R and Hs30R.

Acknowledgement

We thank Dr. Hans J. Knoblich, Medical Director, Bayer Yakuhin Ltd., Tokyo (Japan), for supplying the nifurtimox (Lampit®).

References

1 Cohen, S.M., and G.T. Bryan, Carcinogenesis caused by nitroftLran derivatives, Abstr. 5th Int. Congr., (1972) 23--28.

2 G~nnert , R., Nifur t imox: causal t rea tment of Chagas' disease, Arzneim.-Forsch. (Drug Res.), 22 (1972) 1563.

3 Kondo, S., and H. Ichikawa-Ryo, Testing and classification of mutagenic i ty of furylfuramide in Esche- richia coli, JPn. J. Genet., 4 (1973) 295--300.

4 Kondo, S., H. Ichikawa, K. lwo and T. Kato, Base-change mutagenesis and prophage induct ion in strains of Escherichia col i with different DNA repair capacities, Genetics, 66 (1970) 187--217.

5 McCaila, D.R., and D. Voutsinos, On the mutagenic i ty of nitrof~trans, Mutat ion Res., 26 (1974) 3-- 16.

6 0 h n i s h i , T., S. Yamada, T. Nomura and K. Nozu, Mutagenicity of ant imicrobiai medicine containing acetylfuratr izine, Jpn. J. Genet., 52 (1977) 309--314.

7 Setlow, R.B., Summary of a conference on radiat ion microbiology, Radiat . Res., Suppl., 6 (1966) 220--226.

8 Strauss, B.S., DNA repair mechanisms and their re la t ion to muta t ion and recombinat ion , Current Topics Microbiol. Immunol . , 44 (1968) 1--85.

9 Tazima, Y., Some problems on the methodo logy of chemical mutagenesis in the s i lkworm, Ind. J. Genet. Breed., 34A (1974) 302--310.

10 Tazima, Y., T. Kada and A. Murakami, Mutagenicity of n i t rofuran derivatives, including furylfura- mide, a food preservative, Mutat ion Res., 32 (1975) 55--80.

11 Tonomura, A., and M. Sasaki, Chromosome aberrat ions and DNA repair synthesis in cul tured human cells exposed to ni trofurans, Jpn. J. Genct., 48 (1973) 291--294.

12 Witkin, E.M., Ultraviolet-induced mu ta t ion and DNA repair, Annu. Res. Genet., 3 (1969) 525--552. 13 Witkin, E.M., Ultraviolet mutagenesis inducible DNA repair in Escherichia col i , Bacteriol. Rev., 40

(1976) 869--907.