The mutagenic action of quindoxin, carbadox, olaquindox and some other N-oxides on bacteria and yeast

233

Mutation Research, 78 (1980) 233--242 @)Elsevier/North-Holland Biomedical Press

THE MUTAGENIC ACTION OF QUINDOXIN, CARBADOX, O L A Q U I N D O X AND SOME O T H E R N-OXIDES ON BACTERIA AND YEAST

C.E. VOOGD, J.J. van der STEL and J.J.J.A.A. JACOBS

Laboratory of Chemotherapy, National Institute of Public Health, P.O. Box 1, 3720 BA Bilthoven (The Netherlands) (Received 22 November 1979) (Revision received 6 February 1980) (Accepted 19 February 1980)

Summary The mutagenic action of 3 coccidiostatic chinoxaline
Some chinoxaline di-N-oxide c o m p o u n d s p r o m o t e growth, if added to feed, because of their coccidiostatic action. 3 derivatives are much used for this purpose. Quindoxin or quinoxaline 1,4~lioxide is added to poultry feed at a dosage of 50 mg/kg food (Broome and Bowie, 1972). Carbadox is used in pig

234

food at a concentration of 50 mg/kg. Olaquindox has been added to pig and cattle food in quantities ranging from 25 to 100 mg/kg (Kirchgessner and Roth, 1977; Roth and Kirchgessner, 1977). Quindoxin appears to be carcinogenic after dally administration of 10 mg/kg body weight to rats (Tucker, 1975). Furthermore, this compound can cause contact eczema (Dawson and Scott, 1972). These effects gave rise to this investigation on the mutagenicity of these compounds. To permit comparison with known mutagens, a number of other N-oxides, such as 4-nitroquinoline 1-oxide and quinoline 1-oxide were investigated, as were the mutagenic properties of benzofuroxan (an intermediate in organic chemistry), 4-nitropyridine 1-oxide, pyridine 1-oxide and 4-picoline 1-oxide. A carcinogenic action of 4-nitroquinoline 1-oxide is reported (Paul et al. 1971). Furthermore, this compound is a known mutagenic agent, causing GC--AT transitions, GC--TA transversions and presumably also GC--CG transversions (Prakash et al., 1974). Materials and methods The following substances were provided by the manufacturers, whose generosity is gratefully acknowledged. Quindoxin (No. 2161001-NU 190) by I.C.I., Macclesfield (Great Britain); carbadox (Lot 47237) by Pfizer, Groton (U.S.A.); olaquindox and benzofuroxan by Bayer GmbH, Leverkusen (F.R.G.); dimetridazole by BASF, Ludwigshafen (F.R.G.); furazolidone by Orphahell, Amsterdam (The Netherlands). Quinoline 1-oxide (Lot 062757), pyridine 1-oxide (Lot 122777) and 4-picoline 1-oxide were bought from Aldrich; 4-nitroquinoline 1-oxide (Lot 9516-A) from K and K; and 4-nitropyridine 1-oxide (B 880556) from Fluka. Fluctuation tests (Luria and Delbrtick, 1943) were carried out as described previously (Voogd et al., 1977). As test organisms a Klebsiella pneumoniae mutant, requiring uracil and proline as growth factors, and Escherichia coli K12 Hfr Hayes were used. After incubation during 20 h at 37°C, the total number of streptomycin-resistant (including streptomycin-dependent) bacteria was determined by the pour-plate technique. The average spontaneous mutation rate of Klebsiella pneumoniae was 0.1676 × 10 -9 (S.D. 0.0304 × 10 -9) obtained from 26 independent experiments, and that of E. coli was 0.2393 × 10 -9 (S.D. 0.0596 × 10 -9) obtained from 23 experiments. In addition to this test, the plate-incorporation test developed by Ames (Ames et al., 1975) was used with Salmonella typhimurium TA98 and TA100. These strains were kindly supplied by Professor Ames (Department of Biochemistry, University of California, Berkeley, CA 94720 BC 01, U.S.A.). No metabolic activation was applied. On each plate 3 ml of top agar, in which the compound under investigation was present, were added to 20 ml of the selection medium. The compounds were dissolved in dimethylsulphoxide. To assess the mutagenic effect of quindoxin, carbadox and olaquindox on a eucaryotic organism, Saccharomyces cerevisiae D4 was grown in the presence of these compounds (Zimmermann, 1973). The starting inoculum was 100 yeast cells per ml. After growth during 45 h at 30°C in yeast--peptone medium, the

235 TABLE 1 COMPOUNDS I N V E S T I G A T E D O t Quindoxin or Grofas @ Quinoxaline 1,4-dioxide M.W. 1 6 2 . 1 5

o o t

o ,-c-~N--N---C--OCH3

Carbadox Hydrazineca~boxylic acid- ( 2-quino xolinylmethylene)-methylestez N 1N4-dioxide M.W. 2 6 2 . 2

O O t O N I1 H ~"~--O--N--CI{2--CH 2 O H

~

~-CH3

N 0

Olaquinox, Bayo-N-ox, Bay Va 9391 2-(N-2'-Hydrox yethyt-carbamoyl )-3-methylquinoxaline 1,4-dioxide M.W. 2 6 3 . 3

NO 2 I

<7>

4-Nitroquinoline 1-oxide M.W. 1 9 0 . 1 6

N

O

Quinoline 1 - o x i d e N

M.W. 1 4 5 . 1 6

O O t Benzofttroxan (Bayer 94-812) N/

M.W. 1 3 6 . 0

NO 2 I

O

4-Nitropyridine 1-oxide

N 4 O

©

M.W. 1 4 0 . 1

Pyridine 1-oxide M.W.

N

95.1

O

CH3

© o

4-Picoline 1-oxide M.W. 1 0 9 . 1 3

236 increase in mitotic gene convertants was determined at the highest concentration at which multiplication occurred. Only by this m e t h o d is it possible to demonstrate the mutagenic action of dimetridazole, a mutagenic nitroimidazole, on this yeast strain. Dimetridazole and furazolidone were included because the mutagenic activity of these compounds on micro-organisms is well known. Results In the fluctuation test an increase in the mutation rate of Klebsiella pneumoniae was caused by quindoxin, carbadox, olaquindox, 4-nitroquinoline 1-oxide, quinoline 1-oxide and benzofuroxan (Tables 2 and 3). With 4-nitropyridine 1-oxide, pyridine 1-oxide and 4-picoline 1-oxide no mutagenic action was observed wii~h this organism. A doubling of the mutation rate resulted from the presence of carbadox at a concentration of +5 X 10 -s mmole/1, quindoxin at +10 X 10 -s mmole/1 and

TABLE

2

INCREASE DOX

AND

Substance

IN THE

MUTATION

RATE

OF

Klebsiella p n e u m o n i a e C A U S E D

Carbadox

Olaquindox

CARBA-

Concentration

Number

Cultures

Number

Mutation

Increase

(mmole/1)

bacteria/ml

of

without

mutants

rate

in mutation

after

mutants

in median

(X 10 -9)

rate

20 h

incubation (XIO 9 ) Quindoxin

BY QUINDOXIN,

OLAQUINDOX of

culture

(×)

0.01

0.44

--

90

19.18

116

0.005

0.77

--

84

10.30

63

0.002

1.70

--

116

6.072

37

0.001

1.83

--

50

2.860

17

0.0005

1.83

--

23

1,567

9.5

0.0002

1'.66

--

8

0.793

4.8

0.0001

1.70

9

3

0.401

2.4

0.00005

1.57

29

--

0.219

--

0.00002 0

1.47 1.64

42 51

---

0.164 0.114

---

0.005

0.191

--

10

8.08

49

0.002

0.50

--

49

10.38

63

0.001

0.81

--

34

4.765

29

0.0005

1.11

--

21

2.346

14

0.0002

1.39

--

8

0.949

0.0001

1.43

--

4

0.574

3.5

0,00005

1.38

15

2

0.381

2.3

0.00002

1.37

27

--

0.266

1.6

0.00001

1.61

33

--

0.192

--

0

1.61

46

--

0.01

0.88

5.8

0,134

--

--

47

5.686

35 17

0.005

1.13

--

27

2.873

0.002

1.42

--

10

1.087

6.6

0.001

1.39

--

5

0.687

4.2

0.0005

1.28

12

3

0.461

2.8

0.0002

1.34

19

--

0.344

2.1

0.0001

1.40

32

--

0,225

--

0

1.37

46

--

0.157

--

237 TABLE

3

INCREASE

IN

THE

MUTATION

RATE

OF

Klebsiella pneumoniae

CAUSED

BY SOME

N-OXIDE

COM-

POUNDS

Substance

4-Nitro-

quinoline 1-oxide

Concentration ( m m o l e fl)

1-oxide

Benzofuroxan

4-Nitropyridine 1-oxide

Cultures without mutants

Number of mutants in median cultures

Mutation rate (X 10 -9)

Increase in mutation rate (X)

0.01

1.84

--

4

0.447

0.005

1.75

15

3

0.300

1 .S

0.002

1.69

26

--

0.221

--

O Quinoline

Number of bacteria/ml after 2 0 h incubation (× 10 9 )

2.7

1.24

51

--

0.151

--

20

0.73

42

--

0.332

2.0

10

1.20

26

--

0.310

1.9

5

1.58

19

--

0.292

1.7

2

1.69

22

--

0.249

--

1

1.70

32

--

0.186

--

0

1.57

39

--

0.167

--

1

0.69

--

12

2.706

16

0.5

0.93

--

14

1.893

11

0.2

1.13

--

14

1.739

10.4

0.1

1.08

--

11

1,535

9.2

0.05

1.17

--

6

0.922

5.5

0.02

1.79

3

5

0.532

3.2

0.01

1.71

15

2

0.308

1.8

0.005

1.64

20

--

0.272

1.6

0

1.48

48

--

0.137

--

0.003

1.72

32

--

0.183

--

0.002

1.90

28

--

0.186

--

0.001

1.62

28

--

0.218

--

0.188

--

0

1.39

39

--

Pyridine 1-oxide

0.1

1.67

45

--

0.133

--

4-Picoline

0.02

2.01

29

--

0.171

--

0

1.24

51

--

0.151

1-oxide

olaquindox at -+20X 10 -s mmole/1. In this test, 4-nitroquinoline 1-oxide doubled the mutation rate at a concentration of +0.005 mmole/1, benzofuroxan at +0.01 mmole/1 and quinoline 1-oxide at 20 mmole/1. Therefore, in this test the last 3 substances were less mutagenic than the quinoxaline di-N-oxides. When Escherichia coli K12 was used as a test organism, the quinoxaline N-oxides were also mutagenic (Table 4). Carbadox was more mutagenic than quindoxin. Olaquindox showed the lowest mutagenic activity. The results obtained with Salmonella typhimurium TA100 differed from those obtained with the test organisms mentioned above. Carbadox was mutagenic at a concentration of 0.001 mmole/1 in the top agar, as was olaquindox in the top agar at 0.02 mmole/1 and quindoxin at 0.05 mmole/1 (Table 5). In comparison with the results obtained with the fluctuation test, these quinoxaline di-N-oxides were less mutagenic when applied to Salmonella typhimurium TA100 than with Klebsiella pneumoniae and Escherichia coli K12. On the contrary, 4-nitroquinoline 1-oxide at 0.0001 mmole/1 top agar had a potent mutagenic effect on Salmonella typhimurium TA100 and, in the fluc-

238

TABLE

4

INCREASE

IN THE MUTATION

O F Escherichia coli K 1 2

RATE

CAUSED

BY QUINDOXIN,

CARBA-

DOX AND OLAQUINDOX Concentration (mmole/l)

Quindoxin Mutation rate (× 10 -9 )

Carbadox Increase in mutation rate

Olaquindox

Mutation rate (X 1 0 - 9 )

Increase in mutation rate

(X) 0.02 0.01 0.005

28.87 83.09 27.29

Mutation rate (× 10 -9 )

Increase in mutation rate

(X)

120 347 114

. -6.31

0.002 0.001 0.0005

7.59 3.15 1.§1

31.7 13.2 6.7

28.11 9.38 4.09

0.0002 0.0001 0.00005

0.82 0.69 0.45

3.4 2.9 1.5

1.17 0.69 .

0.00002 0

0.30

.

0.231

--

.

.

(X)

. -26

. 12.04 4.34

50.3 18.1

1.67 0.93 0.40

7.0 3.9 1.7

0.25 0.30

---

0.244

--

117 39 17 4.9 2.9 .

.

. 0.180

.

. .

--

tuation test, doubled the mutation rate of Klebsiella pneumoniae at +0.005 mmole/1. Quinoline 1-oxide had no mutagenic effect on Salmonella typhimurium T A 1 0 0 . A weak mutagenic activity was observed with Salmonella typhimurium T A 1 0 0 when benzofuroxan was present in the top agar at I mmole/1.

TABLE

5

MUTAGENIC ACTION OF SOIvIE QUINOXALINE nella t y p h i m u r i u m T A 1 0 0

N-DIOXIDES

AND

OTHER

N-OXIDES

ON Salmo-

Average number of revertants per plate Concentration in top agar (mmole/1)

Substance

100

50

20

10

5

2

1

0.5

90

130

123

128

n.g. 140 n.g.

n.g. 133 266 *

n.g. 159 197

n.g.

n.g.

n.g.

Quindoxin Carbadox Olaquindox 4-Nitroquinoline 1-oxide a Quinoline 1-oxide Benzofuroxan 4-Nitropyridine 1-oxide Pyridine 1-oxide 4-Pieoline 1-oxide

n.g. 165 137

150 137

The plate-incorporation test was used. To each plate (containing 20 ml of agar) 3 ml top agar were added. The test was done in triplicate. * Mutagenic response. n.g., n o growth. a Number o f r e v e r t a n t s a t 0 . 0 0 0 5 m m o l e f l , 6 0 7 *; 0 . 0 0 0 2 m m o l e / 1 , 3 1 8 * ; 0 . 0 0 0 1 m m o l e / l , 2 1 1 *; 0.00005

mmole/l,

192.

239 In contrast with quinoline 1-oxide and benzofuroxan, 4-nitro-pyridine 1-oxide had no mutagenic effect on Klebsiella pneumoniae, but a mutagenic action was observed when it was tested with Salmonella typhimurium TA100 in the top agar at 0.02 mmole/1. Because this c o m p o u n d inhibits the growth of Klebsiella pneumoniae, the highest concentration investigated by the fluctuation test was 0.003 mmole/1. Pyridine 1-oxide and 4-picoline 1-oxide showed no mutagenic activity when applied to Salmonella typhimurium TA100. Thus, with the exception of pyridine 1-oxide and 4-picoline 1-oxide, all the compounds investigated induced base-pair substitutions. With Salmonella typhimurium TA98, as frame-shift test strain, a mutagenic action was found with the same compounds as with Salmonella typhimurium TA100 (Table 6). Most compounds required higher concentrations for a mutagenic action on TA98 than on TA100. The conclusion is that quindoxin, carbadox, olaquindox, 4-nitroquinoline 1-oxide, benzofuroxan and 4-nitropyridine 1-oxide can induce both base-pair substitutions and frame-shift mutations. To establish a possible mutagenic action of the coccidiostatics on eucaryotic organisms, the mutagenicity of quindoxin, carbadox and olaquindox on growing yeast was investigated by using Saccharomyces cerevisiae D4 (Zimmermann, 1973). From the results in Table 7 it appears that a considerable increase in the number of convertants per yeast cell occurred when these substances were present in the culture medium. The results also suggest that in this system olaquindox is less mutagenic than are quindoxin and carbadox. With the same experimental m e t h o d , it was found that the number of convertants increased if this yeast was cultivated in the presence of furazolidone or dimetridazole.

0.2 2500

0.1 *

n.g. 143 175 2480

797

0.05 *

399

0.02 *

0.01

204

0.005

4300 * 292 *

1830 * 154

677

n.g.

n.g.

n.g.

n.g.

158

*

702

*

376

0 139

n.g. 912 *

1610

0.001

157

n.g. 1900 *

160 *

0.002

*

178

164

*

303

*

2070 *

226

*

1278 *

33

30

35

54

10

33

58

5

44 72 *

2

31 126 *

1

82 *

233 *

49

460 *

0.2

196 *

42

153 * n.g. 395 *

0.1

88 *

n.g.

48 461 * 109 *

0.05

55

n.g.

38 404 * 36

0.02

n.g.

39 121 * 39

0.01

15

48

0.005

A N D O T H E R N - O X I D E S O N Salmonella t y p h i m u r i u m T A 9 8

363 *

38

0.002

The p l a t e - i n c o r p o r a t i o n test was used. To each plate ( c o n t a i n i n g 20 m l o f agar) 3 m l t o p agar were a d d e d . The test was d o n e in t r i p l i c a t e . * Mutagenie response. n.g., n o g r o w t h . a Number of revertants at 0.0005 mmole/1, 85 * ; 0.0002 mmole/1, 49 ; 0.0001 mmole/1, 32.

4-Nitropyridine 1-oxide Pyridine 1-oxide 4-Picoline 1-oxide

100

0.5

N-DIOXIDES

Average number of revertants per plate C o n c e n t r a t i o n in t o p agar ( m m o l e / 1 )

ACTION OF SOME QUINOXALINE

4-Nitr oquinoline 1-oxide a Quinoline 1-oxide Benzofuroxan

Quindoxin Carbadox Olaquindox

Substance

MUTAGENIC

TABLE 6

200

36

0.001

35

0

O

l-O

241 TABLE 7 I N C R E A S E IN M I T O T I C G E N E C O N V E R S I O . N IN S a c c h a r o m y c e s cerevisiae D 4 C A U S E D BY Q U I N O XALINE N-OXIDE DERIVATIVES Substance

Quindoxin

Expt.

I II

Carbadox

I II

Olaquindox

I II

Furazolidone

I II

D i m e t r i d a z ole

I II

Concentration in b r o t h % (w/v)

N u m b e r of cells p e r m l a f t e r 45 h incubation (X 106 )

N u m b e r of c o n v e r t a n t s p e r y e a s t cell; * (X10 - 5 ) tryptophan locus

adenine locus

0.02 0 0.02 0

4.3 42.4 8.2 21.3

86.3 1.26 77.9 1.80

72.8 0.35 78.8 0.92

0.02 0 0.02 0

66.3 107.8 74.3 105

58.6 0.87 71.6 0.82

25.8 0.39 45.0 0.80

0.05 0 0.05 0

16.7 28.5 42.6 29.0

2.31 1.11 13.20 1.21

1.32 0.54 3.74 1.28

0.01 0 0.01 0

12.6 44.9 23.7 59.8

15.7 1.05 16.8 1.02

30.5 0.72 8.9 0.33

0.05 0 0.05 0

20.6 27.9 22.7 10.4

6.15 1.46 42.0 0.60

4.26 0.48 3.24 0.56

* T h e g e o m e t r i c m e a n ( 1 3 i n d e p e n d e n t E x p t s . ) of t h e n u m b e r of c o n v e r t a n t s is: t r y p t o p h a n 1.25 X 10 - s ( c o e f f i c i e n t of v a r i a t i o n , 2 6 . 9 % ) ; a d e n i n e 0 . 5 0 X 10 - s ( c o e f f i c i e n t of v a r i a t i o n , 4 9 . 8 % ) .

Discussion With Klebsiella pneumoniae, the coccidiostatics, quindoxin, carbadox and olaquindox, were more mutagenic than was 4-nitroquinoline 1-oxide, a compound known to have a mutagenic effect on Escherichia coli (Ikenga et al., 1975), yeasts (Prakash et al., 1974) and Neurospora crassa (Ong et al., 1975). However, when Salmonella typhimurium TA98 and TA100 were used as test strains, the reverse was found: with these bacteria, 4-nitroquinoline 1-oxide was more mutagenic than were the 3 coccidiostatics investigated. The coccidiostatics can induce both base-pair substitutions and frame-shift mutations. With Salmonella typhimurium TA100, a base-pair substitution test strain, these compounds were mutagenic at lower concentrations than with strain TA98, a frame-shift test strain. With Salmonella typhimurium TA1537, another frame-shift test strain, still higher concentrations of the quinoxaline di-N-oxides were required to show a mutagenic action (unpublished results). Quinoline 1-oxide had a weak mutagenic effect on Klebsiella pneumoniae. However, no mutagenic activity occurred with the Salmonella typhimurium strains TA98 and TA100, the results being similar to those obtained by Hollstein {1978).

242

Whereas 4-nitropyridine 1-oxide had a mutagenic effect on the Salmonella typhimurium strains TA98 and TA100, it had none on Klebsiella pneumoniae. This compound is a weak carcinogen, according to Nagao and Sugimura (1972). Pyridine 1-oxide and 4-picoline 1-oxide were not mutagenic in our tests. Our results show that not every N-oxide is mutagenic. Furthermore, large differences in the mutability of different test organisms were found. Whether these differences were due to the Salmonella strains used being repair-deficient, whereas Klebsiella pneumoniae is not, remains to be elucidated. Another possibility is differences in metabolism. Quindoxin is reduced to quinoxaline N-oxide by bacteria (Suter et al., 1978), and free radicals arise during this process. On the other hand, 4-nitroquinoline 1-oxide has to be reduced to 4-hydroxy-aminoquinoline 1-oxide before it can cause cancer and presumably mutations (Paul ,et al., 1971). Hence, further investigations are needed to clarify the observed discrepancies in the mutagenic properties of quinoxaline N-di-oxides and thoSe of some other N-oxides with different test strains. References A m e s , B.N., J. M c C a n n a n d E. Y a m a s a k i ( 1 9 7 5 ) M e t h o d s for d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s w i t h the S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y test, M u t a t i o n Res., 31, 3 4 7 - - 3 6 4 . B r o o m e , A.W.J., a n d R . A . Bowie ( 1 9 7 2 ) T h e g r o w t h p r o m o t i n g a c t i v i t y of q u i n o x a l i n e 1 , 4 - d i o x i d e ( q u i n d o x i n ) in y o u n g c h i c k e n (Gallus dornesticus), Res. Vet. Sci., 13, 3 3 0 - - 3 3 3 . D a w s o n , T . A . J . , a n d K.W. S c o t t ( 1 9 7 2 ) C o n t a c t e c z e m a in a g r i c u l t u r a l w o r k e r s , Br. Med. J., III, 4 6 9 - 470. H o l l s t e i n , M., R. T a l c o t t a n d E. 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