Mutagenicities of carbadox and olaquindox — Growth promoters for pigs

49

Mutation Research, 90 (1981) 49--55 Elsevier/North-Holland Biomedical Press

MUTAGENICITIES OF CARBADOX AND OLAQUINDOX -- GROWTH PROMOTERS FOR PIGS

HARUO YOSHIMURA, MASAYUKI NAKAMURA and TETSUO KOEDA

Second Assay Division, National Veterinary Assay Laboratory, 1-15-1 Tokura, Kokubunji-shi, Tokyo 185 (Japan) and KUNIE YOSHIKAWA

Division of Mutagenesis, National Institute of Hygienic Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158 (Japan) (Received 10 September 1980) (Revision received 27 March 1981) (Accepted 21 April 1981)

Summary Carbadox and olaquindox were examined for mutagenicities in the repair tests with Bacillus subtilis (rec assay) and Salmonella typhimurium (uvr assay) and in the reverse mutation test (TA100 and TA98 of S. typhimurium). Both compounds were positive in the rec and uvr assays, and were highly mutagenic for strains TA100 and TA98. Carbadox was about 6 times more mutagenic than olaquindox in the absence of $9 mix. When incubated in $9 mix or bacterial cytosol (BC) mix for various times at 37°C, carbadox was found to lose its mutagenic activities easier than olaquindox. The mutagenicity of carbadox was almost inactivated at 10 min after incubation with $9 mix, but olaquindox still retained its activities even at 20 min. While carbadox required 20 min to be inactivated in BC mix, olaquindox was not completely inactivated even if incubated for 60 min.

Of various quinoxaline 1,4~li-N~xides synthesized up to now, carbadox and olaquindox are commercially available as growth promoters for pigs in many countries. They are also used in the treatment and prevention of porcine infectious diseases caused by diverse bacteria, especially Salmonella, Escherichia coil and Treponema hyodysenteriae (Bertschinger, 1976; Rainier et al., 1973; Williams and Babcock, 1976). On their toxicity, olaquindox was reported to be slightly lower than carbadox in acute toxicity for rats and mice (Bro~. et al., 1979), though there seemed 0165-1218/81/0000--0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press

50 no significant difference between these c o m p o u n d s in the efficacy of growth promoters for pigs and chickens (Bronsch et al., 1975/76; Broz et al., 1979; R o t h and Kirchgessner, 1977). Oud et al. (1979) reported that carbadox was mutagenic in the micronucleus test. Ohta et al. (1980) made a mutagenicity screening on 18 feed additives and found that carbadox was positive for strains T A 1 0 0 and TA98 of S. typhimurium. Negishi et al. (1980) reported the number of revertant colonies induced by carbadox to be several times more abundant than that induced by quinoxaline 1,4-dioxide per pmole for strains T A 1 0 0 and TA98. However, little is k n o w n on the mutagenicity of olaquindox structurally associated with carbadox. In the present paper, we compare the mutagenicity between carbadox and olaquindox in the repair tests and the reverse mutation tests. Data will also be described on these 2 c o m p o u n d s h o w the mutagenic activities persist in the presence of $9 mix and bacterial cytosol, in order to assess their mutagenicities in the organs and the intestinal tracts. Materials and m e t h o d s

Test compounds The structures of carbadox (methyl-3-[2-quinoxalinylmethylene]carbazateN1,N4-dioxide, mol. wt. 262.2, Pfizer) and olaquindox (2-[N-(2-hydroxyethyl)carbamoyl]-3-methylquinoxaline-l,4-dioxide, mol. wt. 263.3, Bayer) are listed in Fig. 1. The former was dissolved in dimethyl sulfoxide and the later in distilled water before use. Repair test Rec assay. Bacillus subtilis H17 (rec*) and M45 (rec-) with and w i t h o u t the ability of recombinational repair in damaged DNA, kindly supplied from Dr. T. Kada of the National Institute of Genetics were used (Kada et al., 1972). The m e t h o d was essentially in the same manner as described b y Slater et al. (1971), except that basal medium used consisted of 1% bacto-tryptone, 0.25% yeast extract, 0.25% NaC1 and 1.5% agar. Each tester strain in exponential phase of growth was added to 0.6% molten soft agar, 2 ml of which were then overlaid to the basal agar plates. A paper disc (10 m m in diameter) impregnated with 0.1 ml of a solution of test c o m p o u n d was placed in the center of each plate. The inhibition zone was compared between the 2 agar plates after incubation at 37°C for 18 h. Difference of 3 mm or more was judged as positive. Uvr assay. This assay was performed using S. typhimurium T A 1 9 7 8 (uvrB*) and TA1538 (uvrB-) with and w i t h o u t excision-repair enzymes (Ames et al., 1973), in a manner like the rec assay. Briefly, Vogel--Bonnet's medium was

? ~, ~'N~

Y ,N

OCH3 0

? o,

L I

A

~%,,./'~ N , " ~ C H 3 0

(a)

(b)

F i g . 1. S t r u c t u r a l f o r m u l a e o f c a x b a d o x (a) and o l a q u i n d o x ( b ) .

.,H

C%-CHaOH

51 solidified by the addition of 2% glucose and 1.5% agar, on which were poured 2 ml of molten soft agar (0.6%) previously inoculated with each tester strain. The soft agar contained 0.1/~M L-histidine and 0.1/~M D-biotin. In repair tests, N.methyl.N'.nitro.N.nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) served as positive control, and kanamycin as negative control.

Reverse mutation test The mutation test was carried out according to the method of Ames et al. (1975), using S. typhimurium TA100 and TA98. Briefly, 0.1 ml of a solution of test compound and 0.5 ml of $9 mix were placed in a sterile tube, to which was added 0.1 ml of bacterial suspension grown at 37°C for 18 h in a shaking incubator. 2 ml of molten soft agar was added and the resultant mixture was poured onto the minimum glucose agar plate. Histidine-independent revertant colonies were enumerated after incubation at 37°C for 48 h. In tubes requiring no metabolic activation was added 0.5 ml of 1/15 M phosphate buffer (pH 7.4) instead of $9 mix. The media used for the mutation test were the same as used in the uvr assay. Preparation o f 89 mix Male Wistar rats weighing about 200 g were pretreated intraperitoneally with Kanechlor 400 dissolved in olive off at a dose of 500 mg per kg of body weight for the induction of microsomal enzymes. The animals were sacrificed by decapitation 5 days later. The liver was excised, washed in saline and homogenized in 2 vol. of 1.15% KCI in a Potter--Elvehjem-type homogenizer: They were centrifuged (9 000 X g at 4°C for 10 rain) and the resultant microsomal supematant ($9 fraction) was stocked at --80°C until use. $9 mix contained 150 #1 $9 fraction, 50 pM sodium phosphate buffer (pH 7.4), 4 pM MgC12, 16.5 pM KC1, 2.5 pM glucose 6-phosphate, 2 pM NADH, 2 pM NADPH and 2.5 pM ATP per 0.5 ml. Effect o f incubation time on mutagenicities of carbadox and olaquindox in $9 mix and bacterial cytosol (BC) mix 0.1 ml of a solution of test compound (5 #g/plate) was mixed with 0.5 ml of $9 mix or BC mix and incubated at 37°C for various times (0--60 min). 0.1 ml of bacterial suspension (TA100) was added to the mixture just before adding molten soft agar and poured onto the minimum glucose agar plate. Preparation o f BC mix E. coli NIHJ was grown in brain-heart infusion broth, shaking at 37°C for 18 h. After centrifugation at 3 000 × g for 30 min, the cultures were washed once in 1/15 M phosphate buffer (pH 7.4) and disrupted with sonic treatment of 20 kHz for 10 rain (Ultrasonic Disruptor, Model UR200P, Tomy Seiko Co., Tokyo). BC obtained was centrifuged (17 000 X g for 20 min at 4°C) and filtersterilized (Brown and Dietrich, 1979). BC mix contained NADPH-generating systems as the same added in $9 mix.

52 TABLE1 REPAIR TESTS OF CARBADOX AND OLAQUINDOX Compounds

B. subtilis

Dose (/~g/disc)

S. t y p h i m u r i u m

Difference of inhibition

M45

H17

TA1538

TA1979

Difference of inhibition

Carbadox

100 10 1

15.0 11.0 4.5

5.0 1.0 0

10.0 10.0 4.5

8.5 2.0 0

4.5 0 0

4.0 2.0 0

Olaquindox

I00 I0 1

14.0 8.5 2.5

4.5 0 0

9.5 8.5 2.5

8.5 2.0 0

5 0 0

3.5 2.0 0

50 1 100

6.5 21.0 9.5

2.5 2.0 9.5

4.0 19.0 0

6.5 2 6

6.5 2 6

0 0 0

Control MNNG MMS Kanamycin

I n h i b i t i o n z o n e i n d i c a t e d in m m .

Results Repair tests with B. subtilis (rec assay) and S. typhimurium (uvr assay) resulted in Table 1. Carbadox and olaquindox were positive in the rec and uvr assays. Difference of inhibition did not markedly vary between 2 compounds for any of the assays. MNNG and MMS were positive in the rec assay, but negative in the uvr assay. Kanamycin was negative in both assays. In the reverse mutation tests, a linear dose--response was recognized on carbadox and olaquindox within the range of their concentrations tested for strains T A 1 0 0 and TA98, as shown in Fig. 2. Carbadox Was more mutagenic than olaquindox with or without the presence of S9 mix and their activities were 3000

Olaquindox

ox ~ 2OOO

2

to ~. 1 0 0 0

uo .~ 1 2 0 0

Carbado× (TA

Olaquindox

98)

( TA 98

)

> 8O0 r~

400

• 215

g

'

~'o

ib

0"2:5

.~'

'

1'o

1:5

Dose ( ug / plate )

Fig. 2. R e s p o n s e o f S. t y p h i m u r i u m w i t h $9 m i x ; o, w i t h o u t $9 m i x .

T A 1 0 0 and T A 9 8 t o various d o s e s o f c a r b a d o x and o l a q u i n d o x , e ,

53

TABLE 2 COMPARISON OF MUTAGENICITY BETWEEN CARBADOX AND OLAQUINDOX TA100

Compounds

Caxbadox Olaquindox

TA98

+$9

--$9

+$9

--$9

146.8 38.3

315.4 46.3

22.2 9.2

68.2 12.3

Values indicate the n u m b e r o f revertant c o l o n i e s induced per #g.

marked with strain TA100. The mutagenicities of these compounds were reduced by the addition of $9 mix. The number of revertant colonies induced by 1 #g of each compound were calculated from the linearity o f the dose-response curve and indicated in Table 2. Carbadox was about 6 times more mutagenic than olaquindox in the absence of $9 mix. By the addition of $9 mix, the number of revertant colonies were more decreased with carbadox than with

olaquindox. The mutagenicities of carbadox and olaquindox were reduced in accordance with incubation time, when allowed to stand in $9 mix or BC mix (Fig. 3). The mutagenicity of carbadox, compared with olaquindox, was easily inactivated in $9 mix, as well as in BC mix. When incubated in $9 mix, carbadox was almost inactivated at 10 min after incubation, but olaquindox still retained its activity after 20 min. While carbadox required 20 min to be inactivated in BC mix, olaquindox was hardly inactivated and not reaching t o spontaneous levels (about 150 colonies/plate) after 60 min. Neither carbadox nor olaquindox lost their mutagenic activities in $9 and BC fractions (without the addition of NADPH-generating systems). 3OOO

200C

Carbadox in S - 9 mix

in BC mix

I(X~

.

8

E 6o0 o o u

Olaquindox

in

Olaquindox

S - 9 mix

in BC mix

~ 4O(

2(x

0 "lb

2"0 3b

6b Incubation

0 "lb

2b 30

6b

time (rain)

Fig. 3. Decrea=e o f mutagenictties o f c a r b a d o x and o l a q u i n d o x in S9 m i x and BC m i x . e , w i t h N A D P H generating s y s t e m s ; X, w i t h o u t NADPH-generating s y s t e m s ; o , p h o s p h a t e b u f f e r as eontroL T A I O 0 w a s used as tester =train.

54 Discussion The rec assay shows positive for chemicals causing UV-induced-like damages and single-strand breaks in cellular DNA (Kada et al., 1972). This assay also responds to chemicals able to cross-link DNA (Kada et al., 1972). In our study, however, the uvr assay did n o t respond to MNNG and MMS, which were reported to cause single-strand breaks in DNA (Kondo et al., 1970). If carbadox and olaquindox are chemicals causing only single-strand breaks in DNA, they must be positive in the rec assay b u t negative in the uvr assay. As both c o m p o u n d s used in this study were, however, positive in the rec and uvr assays, it is assumed that DNA in bacterial cells is damaged by UV-induced-like dimers or cross-links, and n o t by only single-strand breaks. It is known, on the other hand, that such chemicals as mitomycin C able to cross-link DNA do not induce mutations in the uvr- strains and both T A 1 0 0 and TA98 used in this study are uvrB--deficient strains (McCann et al., 1975). From these findings, we conclude that both carbadox and olaquindox induce frameshift and basepair substitution mutations as a result of error in the repair process of D N A which might be damaged by at least UV-induced-like dimers. Carbadox is converted into some metabolites when fed to pigs, the final metabolite being quinoxaline-2-carboxylic acid remaining longest in liver and excreted in urine (Ferrando et al., 1975). Quinoxaline-2-carboxylic acid was non-mutagenic as far as we examined (unpublished data), b u t it is unclear on the mutagenicities of any intermediate metabolites. The mutagenicity of carbad o x was reported to be inactivated gradually in $9 mix (Ohta et al., 1980). In this study, n o t only carbadox b u t also olaquindox were shown to lose their mutagenic activities by NADPH-depending enzymes in $9 mix, as well as in BC mix, especially carbadox being more rapidly inactivated than olaquindox. Although these c o m p o u n d s are anticipated to be converted gradually to nonmutagens in porcine b o d y and also b y some intestinal bacteria, their residues in pigs cannot be ignored on intermediate metabolites which m a y be mutagenic and distributing for a while. If attention is paid to carbadox and olaquindox on the possible hazard to consumers, the use of these c o m p o u n d s should be restricted to become no residues in pigs. Most carcinogens are mutagenic (Ames et al., 1973, 1975; McCann et al., 1975~ Slater et al., 1971). Quinoxaline 1,4-dioxide which had been used as a growth p r o m o t e r for pigs and chickens was withdrawn from the market, because of the formation of nasal and hepatic tumors in rats (Tucker, 1975). Pathological evaluation will also be required to carry o u t on these 2 c o m p o u n d s used in this study. References Ames, B.N., F.D. Lee and W.D. Durston (1973) An improved bacterial test s y s t e m for the d e t e c t i o n and classification o f m u t a g e n s and carcinogens, Proc Natl. A c a d . Sci. (U.S.A.), 70, 782--786. A m e s , B.N., J. McCann a n d E. Yamasaki (1975) M e t h o d s for detecting c a r c i n o g e n s and m u t a g e n s w i t h the S a l m o n e n 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 R e ~ , 31, 347--364. Bertschinger, H.U. (1976) Die c h e m o t h e r a p e u t i s c h e Wirksamkeit yon O l a qui ndox bei Ferkeln m i t experim e n t e l l e r Colidiarrh6e u n d Co]ienterotox//mie, Schweiz. Arch. Tierheilkd., 118, 397--408. Bronsch, K., D. Schneider and F. Rlgal-Antonelli ( 1 9 7 5 / 7 6 ) Ol a qui ndox - - a n e w growth p r o m o t e r in animal nu tritio n, 1. Efficacy in piglet rearing, Z. Tierphysiol. Tierernk~Lrg. F u t t e r m i t t e l k d . , 36, 211--221.

55 Brown, J.P., and P.S. Dietrich (1979) Mutagenicity of anthraqui none and b e n z a n t h r o n c derivatives in the Salmo nella/microso me test: activation of a n t h r a q u i n o n e glycosides by enzymic extracts of rat cecal bacteria, Mu tation Res., 66, 9--24. BroW, J., B. Sev~ik, J. Strakov~, T. Svoboda, J. Nastuneak and J. Hebk~ (1979) C ya dox -- a new s ynt he t i c growth p r o m o t e r of Czechoslovak provenance -- its essential characteristics and effectivity, Biol. Chem. Vet. (Praha), 15, 115--124. Ferrando, R., R. Truhaut, J.-P. R a y n a u d and J.-P. Spanoghe (1975) La toxicit~ de relals, II. A ppl i c a t i on de la m~thodologie "toxicit~ de relais" a l'~valuation de la secvLrit~ d ' e m p l o i pour les consomateurs h u m a i n s du carbadox, facteur de croissance, ajout~ ~ la ration du porc charcutier, Toxicology, 3, 369-396. Kada, T., K. Tutikawa and Y. Sadaie (1972) In vitro host-mediated '~ec assay" procedures for screening chemical mutagens; and phloxine, a m u t a g e n red dye detected, Mutation Res., 16, 165--174. Kondo, S., H. Ichikawa, K. Iwo and T. Kato (1970) Base-change mutagenesis and prophage i n d u c t i o n in strains of Escherichia coli with different DNA repair capacities, Genetics, 66, 187--217. McCann, J., N.E. Spingarn, J. Kobori and B.N. Ames (1975) Detection of carcinogens as mutagens: bacterial tester strains with R factor plasmids, Proc. Natl. Acad. Sci. (U.S.A.), 72, 979--983. Neglshi, T., K. Tanaka and H. Hayatsu (1980) Mutagenicity of carbadox and several quinoxaline 1,4-dioxide derivatives, Chem. Pharrn. Bull., 28, 1347--1349. Ohta, T., M. Moriya, Y. Kaneda, K. Watanabe, T. Miyazawa, F. Sugiyama and Y. Shirasu (1980) Mutagenicity screening of feed additives in the microbial system, Mutation Res., 77, 21--30. Oud, J.L., A.H.H. Reutlinger and J. Branger (1979) An investigation i nt o the cytogenetic damage induced by the coccidiostatic agents amproHum, carbadox, dimetridazolc and ronidazolc, Mut a t i on Res., 68, 179--182. Rainier, R.H., R.R. Chalqucst, W.E. Babcock and G.W. Thrasher (1973) Evaluation of carbadox for prop h y l a x i s and t r e a t m e n t of induced swine dysentery, J. Am. Vet. Med. Ass., 163, 457--461. R o t h , F.X., and M. Kirchgessner (1977) Zur nutritiven Wirkung yon Olaquindox bei Mastschweinen u n d Broflern, Zuchtungskunde, 49, 66--74. Slater, E.E., M.D. Anderson and H.S. Rosenkranz (1971) Rapid d e t e c t i o n of mut a ge ns and carcinogens, Cancer Res., 3 1 , 9 7 0 - - 9 7 3 . Tucker, M.J. (1975) Carcinogenic action of quinoxaline 1,4-dioxide in rats, J. Natl. Cancer Inst., 55, 137--145. Williams, B.J., and W.E. Babcock (1976) In vitro susceptibility of Treponema hy'odysenteriae to carbadox, virginiamycin, and tylosin, Vet. Med. Small Anita. Clln., 7 1 , 9 5 7 - - 9 5 9 .