Chem.-Biol. Interactions, 15 (1976) 69--75 69 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands
MUTAGENIC ACTIVITY OF CARCINOGENIC AND NONCARCINOGENIC N I T R O F U R A N S AND OF U R I N E OF RATS FED THESE COMPOUNDS
CHING YUNG WANG and LANFONG HSU LEE Division of Clinical Oncology, Wisconsin Clinical Cancer Center, Madison, Wisconsin 53706 (U.S.A.) (Received February 16th, 1976) (Revision received April 26th, 1976) (Accepted May 18th, 1976)
SUMMARY
The nitrofurans, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), N-[4-(5nitro-2-furyl)-2-thiazolyl]formamide (FANFT), nitrofurantoin, 5-nitro-2furoic acid, 5-nitro-2-furamidoxime, 5-nitrofurfurylidene diacetate and the urine of rats fed these compounds, were assayed for mutagenic activity in Salmonella typhimurium strains TA100 and T A 1 0 0 F R 1 . All the nitrofurans were mutagenic in the order: AF-2 and F A N F T > nitrofurantoin > 5-nitro2 - f u r a m i d o x i m e > 5-nitrofurfurylidene diacetate > 5-nitro-2-furoic acid. Strain TA100 was more sensitive than T A 1 0 0 F R 1 to the mutagenic influence of these nitrofurans. Only the urine of rats fed AF-2, F A N F T and nitrofurantoin had mutagenic activity. Again, TA100 was more sensitive than T A 1 0 0 F R 1 . The mutagenicity of the urine was not increased by treatment with fl-glucuronidase. AF-2, 2-amino-4-(5-nitro-2-furyl)thiazole (deformylated product of F A N F T ) and nitrofurantoin were excreted in the urine of rats fed these compounds; whereas the other nitrofurans were not excreted.
INTRODUCTION
Nitroreduction has been linked to the mutagenicity [ 1,2] and carcinogenicity [3] of nitroheterocycles. Nitroreduction of these compounds has been catalyzed by several mammalian enzymes [4--8]. All the nitroheterocycles tested thus far exhibited mutagenic activity in microorganisms [1,9--11], however, those nitroheterocycles with short chain substitutions to the he-
Abbreviations: AF-2, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; ANFT, 2-amin0-4-(5-nitro2-furyl)thiazole; FANFT, N-[4-(5-nitro-2-furyl)-2-thiazotyl]formamide.
70 terocyclic nuclei were generally weak or not carconogenic [12]. The failure of these nitroheterocycles to induce tumors may be due to their weak mutagenic activity [1] or weak ability to induce DNA damage [13]. It is also possible that these nitroheterocycles were detoxified in vivo at a faster rate and lost the ability to induce tumors. To test the latter hypothesis, we investigated the mutagenesis of urine of rats fed carcinogenic and noncarcinogenic nitrofurans and the excretion of these compounds in the urine. FANFT and AF-2 have been shown to be carcinogens [9]. 5-Nitro-2furoic acid [9], 5-nitro-2-furamidoxime [12] and 5-nitrofurfurylidene diacetate [3] are not carcinogenic in rats. Nitrofurantoin, widely used in the treatm e n t of human urinary tract infections, was also not carcinogenic in rats [3,12]. Because nitrofurantoin demonstrated mutagenic activity in E. coli [11] and S. t y p h i m u r i u m [2,10], it was suspected of being a carcinogen [2,11]. MATERIALS AND METHODS Chemicals. FANFT and 5-nitro-2-furamidoxime were obtained from Abbott Laboratories, North Chicago, Ill.; 5-nitro-2-furoic acid and 5-nitrofurfurylidene diacetate from Aldrich Chemical Co., Milwaukee, Wis.; and nitrofurantoin from Eaton Laboratories, Norwich, N.Y. ANFT was prepared as described [6]. Bacterial ~-glucuronidase was obtained from Sigma Chemical Co., St. Louis, Mo. Animal experiments. Female Sprague-Dawley rats, 150--200 g, were obtained from Sprague-Dawley, Madison, Wis. The animals were randomly divided into 7 groups, 2 animals per group, and housed in screen-bottom metal cages. Powdered Wayne Lab-Blox, Allied Mills, Chicago, Ill. was the basal diet fed the control group of rats. The experimental groups were given a basal diet containing 0.5% AF-2, FANFT, nitrofurantoin, 5-nitro-2-furoic acid, 5-nitro-2-furamidoxime, or 5-nitrofurfurylidene diacetate. The animals received the diets and water ad libitum for 4 days. On the 5th day, the pairs of rats were housed in metal metabolic cages and received water only. The water bottle was placed outside the cage and within reach of the rats so that water drained from the bottle would not mix with urine. The urine excreted in 10 h was collected in a test tube submerged in an ice-bath. The urine was centrifuged, the clear supernatant fluid filtered through a 0.22 pm Millipore filter, and immediately used for the m u t a t i o n test. Mutation assays. The mutation activities of nitrofurans and urine of rats fed nitrofurans were assayed in S. t y p h i m u r i u m strains TA100 [14] and TA100FR1 [2] as described by Ames [15]. The inoculates were grown in Bactonutrient broth (Difco, Detroit, Mich.) at 37°C overnight in the dark. Saline (0.9% NaC1) diluted urine, 0.2 ml, or 25 pl of nitrofuran in dimethyl sulfoxide was added to 2 ml of top agar [15] along with 0.3 ml of the bacterial suspension, 3 to 6 • 107 cells for TA100 or 3 to 6 ' 10 s cells for TA100FR1, and plated. The his ÷revertant colonies were enumerated after incubation in the dark for 3 days at 37°C. The effect of/3-glucuronidase on the
71 m u t a g e n i c i t y o f urine o f rats fed n i t r o f u r a n s was c o n d u c t e d as d e s c r i b e d b y Durston and Ames [16].
Detection o f nitrofurans in urine by thin-layer chromatography (TLC). O n l y t h e urine o f rats fed n i t r o f u r a n t o i n a n d 5-nitro-2-furoic acid was adjusted to b e l o w p H 2 w i t h 2 N HCt b e f o r e e x t r a c t i o n . I m l o f urine was e x t r a c t ed w i t h 4 m l o f n i t r o m e t h a n . N i t r o m e t h a n r e m o v e d a l m o s t all o f t h e nitrof u r a n s in t h e urine. T h e n i t r o m e t h a n e x t r a c t was dried u n d e r an N2 s t r e a m , dissolved in a f e w d r o p s o f e t h a n o l a n d s p o t t e d on a cellulose p l a t e (Polyg r a m C E L 3 0 0 UV2s4, B r i n k m a n I n s t r u m e n t s , Inc., W e s t b u r y , N.Y.). T h e a s c e n d i n g T L C was p e r f o r m e d w i t h an a q u e o u s s o l v e n t o f 2% f o r m i c acid [ 1 7 ] . T h e c h r o m a t o g r a m was e x a m i n e d u n d e r U V light, This m e t h o d c o u l d d e t e c t a level as l o w as 5 p g o f n i t r o f u r a n s . RESULTS AND DISCUSSION T h e m u t a g e n i c a c t i v i t y o f n i t r o h e t e r o c y c l e s has b e e n s h o w n in m i c r o o r g a n i s m s [ 1 , 9 - - 1 1 ] . Strains w h i c h lack t h e ability t o r e d u c e n i t r o g r o u p s u n d e r a e r o b i c c o n d i t i o n s are m u c h less sensitive t o m u t a g e n e s i s b y n i t r o f u r a n s t h a n strains w h i c h h a v e t h e ability t o r e d u c e n i t r o f u r a n s u n d e r a e r o b i c c o n d i t i o n s
TABLE I MUTAGENICITY OF NITROFURANS IN S. typhimurium STRAINS TA100 AND TA100FR1 Nitrofuran
t~g/plate
Number of revertant colonies/ plate a TA100
Control AF-2
78 0.01 0.01
108
502
0.1 Nitrofurantoin 5-Nitro-2-furoic acid
5-Nitro-2-furamidoxime 5-Nitrofurfurylidene diacetate
a Average of duplicate determinations. b Severe growth inhibition was observed.
0.3 3 1 10 100 0.1 1 0.1 1 10
18
660
0.I
FANFT
TA100FR1
72 765 52 68 75 136 5 95 606 78 72 168 b
24 28 22 18 67 27 117
72
[1,2]. Thus, the mutagenic activity seems to be due to the reduced metabolites of these c o m p o u n d s [1,2]. The spontaneous mutation frequency was 4/106 survivals in strain TA100 and 4/10 s survivals in strain T A 1 0 0 F R 1 . Strain TA100 was more sensitive than T A 1 0 0 F R 1 to the mutagenicity of nitrofurans {Table I) because the latter strain lacked aerobic nitroreductase [2]. The mutagenic activity of the nitrofurans tested was: AF-2 and F A N F T > nitrofurantoin > 5-nitro-2-furamidoxime > 5-nitrofurfurylidene diacetate > 5-nitro-2-furoic acid {Table I). The mutagenicity of the urine of rats fed these nitrofurans is shown in Table II. Only urine of rats fed AF-2, F A N F T or nitrofurantoin was mutaT A B L E II M U T A G E N I C I T Y O F T H E U R I N E O F R A T S F E D N I T R O F U R A N S IN S. typhimurium STRAINS TA100 AND TA100FR1 Nitrofuran a
Urine b (pl/plate)
N u m b e r of r e v e r t a n t c o l o n i e s / plate c TA100
Control
TA100FR1
78
12
Control
20 2 0.2
91 68 60
10 7 11
AF-2
20 2 0.2
235 d 132 65
21 8 9
FANFT
20 2 0.2
178 d 87 d 130
53 18 8
Nitrofurantoin
20 2 0.2
426 73
23 13 10
5-Nitro-2-furoic acid
20 2 0.2
100 72 60
11 Ii 10
5-Nitro-2-furamidoxime
20 2 0.2
92 74 63
9 10 12
5-Nitrofurfurylidene diaeetate
20 2 0.2
105 83 82
9 8 11
a F e m a l e Sprague-Dawley rats, 1 5 0 - - 2 0 0 g, were few 0.5% n i t r o f u r a n diets for 4 days. U r i n e was collected d u r i n g t h e first 10 h of t h e 5 t h day. b U r i n e was c e n t r i f u g e d a n d filtered t h r o u g h a n 0 . 2 2 p m Miltipore filter a n d d i l u t e d w i t h sterile saline. E a c h plate c o n t a i n e d 0.2 m l of t h e urine s o l u t i o n . c Average of d u p l i c a t e d e t e r m i n a t i o n s . d Severe g r o w t h i n h i b i t i o n b y u r i n e samples was observed.
73 genic. Again, strain T A 1 0 0 was m o r e sensitive t h a n T A 1 0 0 F R 1 . Unlike the urine o f rats fed N - 2 - f l u o r e n y l a c e t a m i d e , the m u t a g e n i c activity o f which c o u l d be e n h a n c e d b y t h e a d d i t i o n o f fl-glucuronidase t o t h e plate [ 1 6 ] , t h e m u t a g e n i c activity o f urine o f rats fed n i t r o f u r a n s could n o t be e n h a n c e d b y the a d d i t i o n o f / 3 - g l u c u r o n i d a s e (Table III), suggesting t h a t the rats d o n o t e x c r e t e g l u c u r o n i d e c o n j u g a t e d active m e t a b o l i t e s o f n i t r o f u r a n s in t h e urine. This finding was also s u p p o r t e d b y o u r previous d a t a t h a t liver h o m o g e n a t e s r e d u c e d n i t r o f u r a n to a m i n o f u r a n w i t h o u t p r o d u c i n g d e t e c t a b l e nitroso or h y d r o x y l a m i n e [ 1 8 ] . I n c i d e n t a l l y , a m i n o h e t e r o c y c l e s were n o t m u t a g e n i c [ 1 9 ] . T h e r e f o r e , t h e m u t a g e n i c activity o f the urine o f rats fed AF-2, F A N F T o r n i t r o f u r a n t o i n m a y be m a i n l y d u e t o t h e e x c r e t e d n i t r o f u r a n . I n d e e d , as s h o w n in Table IV, AF-2, A N F T and n i t r o f u r a n t o i n were det e c t e d i n ' t h e urine whereas t h e o t h e r n i t r o f u r a n s were not. F A N F T was def o r m y l a t e d b y a r y l f o r m a m i d a s e in several organs o f the rats [17] and exc r e t e d as A N F T in t h e urine. N o F A N F T was d e t e c t e d in the urine. A N F T was m u t a g e n i c in T A 1 0 0 [ 1 0 ] . T h e results d e m o n s t r a t e d t h a t 5-nitro-2furoic acid, 5 - n i t r o - 2 - f u r a m i d o x i m e and 5 - n i t r o f u r f u r y l i d e n e d i a c e t a t e w e r e m e t a b o l i z e d t o such an e x t e n t t h a t n o n e o f these c o m p o u n d s c o u l d be d e t e c t e d in the urine. F A N F T i n d u c e d u r i n a r y b l a d d e r c a n c e r in rats [19] and dogs [ 2 0 ] . T h e u r o t h e l i a l cells o f rat and dog c o u l d activate A N F T , the u r i n a r y m e t a b o l i t e o f F A N F T , and p r o d u c e m a c r o m o l e c u l e - b o u n d a d d u c t s ( u n p u b l i s h e d data). H a s h i m o t o and Kitagawa [21] f o u n d t h a t rat u r o t h e l i a l cells could be transf o r m e d b y N - b u t y l - N - ( 4 - h y d r o x y b u t y l ) n i t r o s a m i n e and N-butyl-N-(3-carb o x y p r o p y l ) n i t r o s a m i n e . T h e s e d a t a i n d i c a t e d t h a t chemical carcinogens
TABLE III EFFECT OF fl-GLUCURONIDASE ON THE MUTAGENIC ACTIVITY OF URINE OF RATS FED NITROFURANS Nitrofuran
Control AF-2 FANFT Nitrofurantoin 5-Nitro-2-furoic acid 5-Nitro-2-furamidoxime 5-Nitrofurfurylidene diacetate
Urine (pl/plate) a
2 2 0.2 2 0.2 20 20 20
Number of revertant colonies/plate (TA100) b Without enzyme
With enzyme c
96 170 508 92 602 108 165 138 95
82 168 516 102 715 125 165 144 139
a Urine was diluted in saline and 0.2 ml was added in each plate. b Average of duplicate determinations. c Each plate contained 5 mg of bacterial 13-glucuronidase.
74 TABLE IV DETECTION OF NITROFURANS IN THE URINE OF RATS Nitrofuran
R[ value on
Color b
Detected in urine
cellulose TLC a
AF-2 FANFT c ANFT c Nitrofurantoin 5-Nitro-2-fuvoic acid 5-Nitro-2-furamidoxime 5-Nitrofurfurylidene diacetate
0.33 0.14 0.23 0.55 0.88 0.63 0.45 d
Visible light
UV light
Y Y Y Y none Y none
DO Y DO Y D D D
Yes
No Yes Yes No No No
a Ascending chromatography with 2% formic acid solvent system. b y ; yellow; DO, dull orange; D, dark; none, invisible.
c Rat fed FANFT and only ANFT was detected in the urine. d Severe tailing on TLC. c o u l d be a c t i v a t e d in susceptible organs. T h e r e f o r e , c a r c i n o g e n i c i t y m a y be a f u n c t i o n o f t h e c o n c e n t r a t i o n o f t h e c a r c i n o g e n in t h e susceptible organ, and m a y explain w h y A F - 2 and F A N F T , w h i c h are m e t a b o l i z e d slowly, are carcinogens, w h e r e a s 5-nitro-2-furoic acid, 5 - n i t r o - 2 - f u r a m i d o x i m e and 5-nitrof u r f u r y l i d e n e d i a c e t a t e , w h i c h are m e t a b o l i z e d r a p i d l y , are n o n c a r c i n o g e n s . T h e failure of these n i t r o f u r a n s to i n d u c e t u m o r s m a y also be d u e to t h e i r w e a k m u t a g e n i c activity [1] or w e a k ability t o i n d u c e D N A d a m a g e [ 1 3 ] . N i t r o f u r a n t o i n has b e e n s h o w n to be n o n c a r c i n o g e n i c in rats [ 3 , 1 2 ] ; h o w ever, this c o m p o u n d has n o t b e e n t e s t e d u n d e r o p t i m a l c o n d i t i o n s [12] and t h e results h a v e b e e n c o n s i d e r e d inconclusive [ 1 1 ] . Since n i t r o f u r a n t o i n was m u t a g e n i c a n d was m e t a b o l i z e d at a slow rate, it m a y be a carcinogen. T h e urine o f m i c e fed F A N F T has b e e n s h o w n to be m u t a g e n i c to E. coli [ 2 2 ] . ACKNOWLEDGEMENTS We are grateful t o Dr. B.N. A m e s , U n i v e r s i t y o f California, B e r k e l e y , California, f o r p r o v i d i n g S. typhimurium strain T A 1 0 0 , Dr. H.S. R o s e n k r a n z , C o l u m b i a U n i v e r s i t y , N e w Y o r k , N e w Y o r k , f o r strain T A 1 0 0 F R 1 , and Dr. T. M a t s u s h i m a , U n i v e r s i t y o f T o k y o , T o k y o , J a p a n , f o r AF-2. We t h a n k Dr. G.T. B r y a n f o r m a n y h e l p f u l criticisms, and K. D e i g h t o n a n d D. S c o t t f o r t h e p r e p a r a t i o n of t h e m a n u s c r i p t . This s t u d y was s u p p o r t e d in p a r t b y U S P H S research grant C A 1 7 4 4 9 f r o m t h e N a t i o n a l C a n c e r I n s t i t u t e t h r o u g h t h e N a t i o n a l Bladder Cancer P r o g r a m . REFERENCES 1 D.R. McCalla and D. Voutsinos, On the mutagenicity of nitrofurans, Mutation Res., 26 (1974) 3.
75 2 H.S. Rosenkranz and W.T. Speck, Microsomal activation of nitrofurantoin to a mutagen, Biochem. Pharmacol., in press. 3 J.E. Morris, J.M. Price, J.J. Lalich and R.J. Stein, The carcinogenic activity of some 5nitrofuran derivatives in the rat, Cancer Res., 29 (1969) 2145. 4 D.R. Feller, M. Morita and J.R. Gillette, Enzymatic reduction of niridazole by rat liver microsomes, Biochem. Pharmacol., 20 (1971) 203. 5 H.E. Paul, V.R. Ells, F. Kopko and R.C. Bender, Metabolic degradation of the nitrofurans, J. Med. Pharm. Chem., 2 (1960) 563. 6 C.Y. Wang, B.C. Behrens, M. Ichikawa and G.T. Bryan, Nitroreduction of 5-nitrofuran derivatives by rat liver xanthine oxidase and reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase, Biochem. Pharmacol., 23 (1974) 3395. 7 C.Y. Wang, C.W. Chiu and G.T. Bryan, Nitroreduction of carcinogenic 5-nitrothiophenes by rat tissues, Biochem. Pharmacol., 24 (1975) 1563. 8 M.K. Wolp~rt, J.R. Althaus and D.G. Johns, Nitroreductase activity of mammalian liver aldehyde oxidase, J. Pharmacol. Exptl. Therap., 185 (1973) 202. 9 C.Y. Wang, K. Muraoka and G.T. Bryan, MutageniciLy of nitrofurans, nitrothiophenes, nitropyrroles, nitroimidazole, aminothiophenes and aminothiazoles in Salmonella typhimurium, Cancer Res., 35 (1975) 3611. 10 T. Yahagi, T. Matsushima, M. Nagao, Y. Seino, T. Sugimura and G.T. Bryan, Mutagenicity of nitrofuran derivatives on a bacterial tester strain with an R Factor plasmid, Mutation Res., 40 (1976) 9. 11 T. Yahagi, M. Nagao, K. Hara, T. Matsushima, T. Sugimura and G.T. Bryan, Relationships between the carcinogenic and mutagenic or DNA-modifying effects of nitrofuran derivatives, including 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide,a food additive, Cancer Res., 34 (1974) 2266. 12 S.M. Cohen, E. Ertiirk, A.M. Von Esch, A.J. Crovetti and G.T. Bryan, Carcinogenicity of 5-nitrofurans, 5-nitroimidazoles, 4-nitrobenzenes, and related compounds, J. Natl. Cancer Inst., 51 (1973) 403. 13 Y. Tu and D.R. McCalla, Effect of activated nitrofurans on DNA, Biochim. Biophys. Acta, 402 (1975) 142. 14 J. McCann, N.E. Spingarn, J. Kobori and B.N. Ames, Detection of carcinogens as mutagens: Bacterial tester strains with R factor plasmids, Proc. Natl. Acad. Sci. USA, 72 (1975) 979. 15 B.N. Ames, The detection of chemical mutagens with enteric bacteria, In A. Hollaender (Ed.), Chemical Mutagens: Principle and Methods for Their Detection, Vol. 1, Plenum, New York, 1971, pp. 267--282. 16 W.E. Durston and B.N. Ames, A simple method for the detection of mutagens in urine: Studies with the carcinogen 2-acetylaminofluorene, Proc. Natl. Acad. Sci. USA, 71 (1974) 737. 17 C.Y. Wang and G.T. Bryan, Deacylation of carcinogenic 5-nitrofuran derivatives by mammalian tissues, Chem.-Biol. Interact., 9 (1974) 423. 18 C.Y. Wang, C.W. Chiu, B. Kaiman and G.T. Bryan, Identification of 2-methyl-4-(5amino-2-furyl)thiazole as the reduced metabolite of 2-methyl-4-(5-nitro-2-furyl)thiazole, Biochem. Pharmacol., 24 (1975) 291. 19 E. Ertfirk, J.M. Price, J.E. Morris, S.M. Cohen, R.S. Leith, A.M. Von Esch, and A.J. Crovetti, The production of carcinoma of the urinary bladder in rats by feeding N[ 4-(5-nitro-2-furyl)-2-thiazolyl ] formamide, Cancer Res., 27 ( 1967 ) 1998. 20 E. Ertiirk, S.A. Atassi, O. Yoshida, S.M. Cohen, J.M. Price and G.T. Bryan, Comparative urinary and gallbladder carcinogenicity of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide and N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide in the dog, J. Natl. Cancer Inst., 45 (1970) 535. 21 Y. Hashimoto and H.S. Kitagawa, In vitro neoplastic transformation of epithelial cells of rat urinary bladder by nitrosamines, Nature, 252 (1974) 497. 22 F.H. Mukai, I. Hawryluk and T.T. Finkelstein, Mutagenic activity in the urine of mice fed FANFT, Mutation Res., 31 (1975) 330.
MUTAGENIC ACTIVITY OF CARCINOGENIC AND NONCARCINOGENIC N I T R O F U R A N S AND OF U R I N E OF RATS FED THESE COMPOUNDS
CHING YUNG WANG and LANFONG HSU LEE Division of Clinical Oncology, Wisconsin Clinical Cancer Center, Madison, Wisconsin 53706 (U.S.A.) (Received February 16th, 1976) (Revision received April 26th, 1976) (Accepted May 18th, 1976)
SUMMARY
The nitrofurans, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), N-[4-(5nitro-2-furyl)-2-thiazolyl]formamide (FANFT), nitrofurantoin, 5-nitro-2furoic acid, 5-nitro-2-furamidoxime, 5-nitrofurfurylidene diacetate and the urine of rats fed these compounds, were assayed for mutagenic activity in Salmonella typhimurium strains TA100 and T A 1 0 0 F R 1 . All the nitrofurans were mutagenic in the order: AF-2 and F A N F T > nitrofurantoin > 5-nitro2 - f u r a m i d o x i m e > 5-nitrofurfurylidene diacetate > 5-nitro-2-furoic acid. Strain TA100 was more sensitive than T A 1 0 0 F R 1 to the mutagenic influence of these nitrofurans. Only the urine of rats fed AF-2, F A N F T and nitrofurantoin had mutagenic activity. Again, TA100 was more sensitive than T A 1 0 0 F R 1 . The mutagenicity of the urine was not increased by treatment with fl-glucuronidase. AF-2, 2-amino-4-(5-nitro-2-furyl)thiazole (deformylated product of F A N F T ) and nitrofurantoin were excreted in the urine of rats fed these compounds; whereas the other nitrofurans were not excreted.
INTRODUCTION
Nitroreduction has been linked to the mutagenicity [ 1,2] and carcinogenicity [3] of nitroheterocycles. Nitroreduction of these compounds has been catalyzed by several mammalian enzymes [4--8]. All the nitroheterocycles tested thus far exhibited mutagenic activity in microorganisms [1,9--11], however, those nitroheterocycles with short chain substitutions to the he-
Abbreviations: AF-2, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; ANFT, 2-amin0-4-(5-nitro2-furyl)thiazole; FANFT, N-[4-(5-nitro-2-furyl)-2-thiazotyl]formamide.
70 terocyclic nuclei were generally weak or not carconogenic [12]. The failure of these nitroheterocycles to induce tumors may be due to their weak mutagenic activity [1] or weak ability to induce DNA damage [13]. It is also possible that these nitroheterocycles were detoxified in vivo at a faster rate and lost the ability to induce tumors. To test the latter hypothesis, we investigated the mutagenesis of urine of rats fed carcinogenic and noncarcinogenic nitrofurans and the excretion of these compounds in the urine. FANFT and AF-2 have been shown to be carcinogens [9]. 5-Nitro-2furoic acid [9], 5-nitro-2-furamidoxime [12] and 5-nitrofurfurylidene diacetate [3] are not carcinogenic in rats. Nitrofurantoin, widely used in the treatm e n t of human urinary tract infections, was also not carcinogenic in rats [3,12]. Because nitrofurantoin demonstrated mutagenic activity in E. coli [11] and S. t y p h i m u r i u m [2,10], it was suspected of being a carcinogen [2,11]. MATERIALS AND METHODS Chemicals. FANFT and 5-nitro-2-furamidoxime were obtained from Abbott Laboratories, North Chicago, Ill.; 5-nitro-2-furoic acid and 5-nitrofurfurylidene diacetate from Aldrich Chemical Co., Milwaukee, Wis.; and nitrofurantoin from Eaton Laboratories, Norwich, N.Y. ANFT was prepared as described [6]. Bacterial ~-glucuronidase was obtained from Sigma Chemical Co., St. Louis, Mo. Animal experiments. Female Sprague-Dawley rats, 150--200 g, were obtained from Sprague-Dawley, Madison, Wis. The animals were randomly divided into 7 groups, 2 animals per group, and housed in screen-bottom metal cages. Powdered Wayne Lab-Blox, Allied Mills, Chicago, Ill. was the basal diet fed the control group of rats. The experimental groups were given a basal diet containing 0.5% AF-2, FANFT, nitrofurantoin, 5-nitro-2-furoic acid, 5-nitro-2-furamidoxime, or 5-nitrofurfurylidene diacetate. The animals received the diets and water ad libitum for 4 days. On the 5th day, the pairs of rats were housed in metal metabolic cages and received water only. The water bottle was placed outside the cage and within reach of the rats so that water drained from the bottle would not mix with urine. The urine excreted in 10 h was collected in a test tube submerged in an ice-bath. The urine was centrifuged, the clear supernatant fluid filtered through a 0.22 pm Millipore filter, and immediately used for the m u t a t i o n test. Mutation assays. The mutation activities of nitrofurans and urine of rats fed nitrofurans were assayed in S. t y p h i m u r i u m strains TA100 [14] and TA100FR1 [2] as described by Ames [15]. The inoculates were grown in Bactonutrient broth (Difco, Detroit, Mich.) at 37°C overnight in the dark. Saline (0.9% NaC1) diluted urine, 0.2 ml, or 25 pl of nitrofuran in dimethyl sulfoxide was added to 2 ml of top agar [15] along with 0.3 ml of the bacterial suspension, 3 to 6 • 107 cells for TA100 or 3 to 6 ' 10 s cells for TA100FR1, and plated. The his ÷revertant colonies were enumerated after incubation in the dark for 3 days at 37°C. The effect of/3-glucuronidase on the
71 m u t a g e n i c i t y o f urine o f rats fed n i t r o f u r a n s was c o n d u c t e d as d e s c r i b e d b y Durston and Ames [16].
Detection o f nitrofurans in urine by thin-layer chromatography (TLC). O n l y t h e urine o f rats fed n i t r o f u r a n t o i n a n d 5-nitro-2-furoic acid was adjusted to b e l o w p H 2 w i t h 2 N HCt b e f o r e e x t r a c t i o n . I m l o f urine was e x t r a c t ed w i t h 4 m l o f n i t r o m e t h a n . N i t r o m e t h a n r e m o v e d a l m o s t all o f t h e nitrof u r a n s in t h e urine. T h e n i t r o m e t h a n e x t r a c t was dried u n d e r an N2 s t r e a m , dissolved in a f e w d r o p s o f e t h a n o l a n d s p o t t e d on a cellulose p l a t e (Polyg r a m C E L 3 0 0 UV2s4, B r i n k m a n I n s t r u m e n t s , Inc., W e s t b u r y , N.Y.). T h e a s c e n d i n g T L C was p e r f o r m e d w i t h an a q u e o u s s o l v e n t o f 2% f o r m i c acid [ 1 7 ] . T h e c h r o m a t o g r a m was e x a m i n e d u n d e r U V light, This m e t h o d c o u l d d e t e c t a level as l o w as 5 p g o f n i t r o f u r a n s . RESULTS AND DISCUSSION T h e m u t a g e n i c a c t i v i t y o f n i t r o h e t e r o c y c l e s has b e e n s h o w n in m i c r o o r g a n i s m s [ 1 , 9 - - 1 1 ] . Strains w h i c h lack t h e ability t o r e d u c e n i t r o g r o u p s u n d e r a e r o b i c c o n d i t i o n s are m u c h less sensitive t o m u t a g e n e s i s b y n i t r o f u r a n s t h a n strains w h i c h h a v e t h e ability t o r e d u c e n i t r o f u r a n s u n d e r a e r o b i c c o n d i t i o n s
TABLE I MUTAGENICITY OF NITROFURANS IN S. typhimurium STRAINS TA100 AND TA100FR1 Nitrofuran
t~g/plate
Number of revertant colonies/ plate a TA100
Control AF-2
78 0.01 0.01
108
502
0.1 Nitrofurantoin 5-Nitro-2-furoic acid
5-Nitro-2-furamidoxime 5-Nitrofurfurylidene diacetate
a Average of duplicate determinations. b Severe growth inhibition was observed.
0.3 3 1 10 100 0.1 1 0.1 1 10
18
660
0.I
FANFT
TA100FR1
72 765 52 68 75 136 5 95 606 78 72 168 b
24 28 22 18 67 27 117
72
[1,2]. Thus, the mutagenic activity seems to be due to the reduced metabolites of these c o m p o u n d s [1,2]. The spontaneous mutation frequency was 4/106 survivals in strain TA100 and 4/10 s survivals in strain T A 1 0 0 F R 1 . Strain TA100 was more sensitive than T A 1 0 0 F R 1 to the mutagenicity of nitrofurans {Table I) because the latter strain lacked aerobic nitroreductase [2]. The mutagenic activity of the nitrofurans tested was: AF-2 and F A N F T > nitrofurantoin > 5-nitro-2-furamidoxime > 5-nitrofurfurylidene diacetate > 5-nitro-2-furoic acid {Table I). The mutagenicity of the urine of rats fed these nitrofurans is shown in Table II. Only urine of rats fed AF-2, F A N F T or nitrofurantoin was mutaT A B L E II M U T A G E N I C I T Y O F T H E U R I N E O F R A T S F E D N I T R O F U R A N S IN S. typhimurium STRAINS TA100 AND TA100FR1 Nitrofuran a
Urine b (pl/plate)
N u m b e r of r e v e r t a n t c o l o n i e s / plate c TA100
Control
TA100FR1
78
12
Control
20 2 0.2
91 68 60
10 7 11
AF-2
20 2 0.2
235 d 132 65
21 8 9
FANFT
20 2 0.2
178 d 87 d 130
53 18 8
Nitrofurantoin
20 2 0.2
426 73
23 13 10
5-Nitro-2-furoic acid
20 2 0.2
100 72 60
11 Ii 10
5-Nitro-2-furamidoxime
20 2 0.2
92 74 63
9 10 12
5-Nitrofurfurylidene diaeetate
20 2 0.2
105 83 82
9 8 11
a F e m a l e Sprague-Dawley rats, 1 5 0 - - 2 0 0 g, were few 0.5% n i t r o f u r a n diets for 4 days. U r i n e was collected d u r i n g t h e first 10 h of t h e 5 t h day. b U r i n e was c e n t r i f u g e d a n d filtered t h r o u g h a n 0 . 2 2 p m Miltipore filter a n d d i l u t e d w i t h sterile saline. E a c h plate c o n t a i n e d 0.2 m l of t h e urine s o l u t i o n . c Average of d u p l i c a t e d e t e r m i n a t i o n s . d Severe g r o w t h i n h i b i t i o n b y u r i n e samples was observed.
73 genic. Again, strain T A 1 0 0 was m o r e sensitive t h a n T A 1 0 0 F R 1 . Unlike the urine o f rats fed N - 2 - f l u o r e n y l a c e t a m i d e , the m u t a g e n i c activity o f which c o u l d be e n h a n c e d b y t h e a d d i t i o n o f fl-glucuronidase t o t h e plate [ 1 6 ] , t h e m u t a g e n i c activity o f urine o f rats fed n i t r o f u r a n s could n o t be e n h a n c e d b y the a d d i t i o n o f / 3 - g l u c u r o n i d a s e (Table III), suggesting t h a t the rats d o n o t e x c r e t e g l u c u r o n i d e c o n j u g a t e d active m e t a b o l i t e s o f n i t r o f u r a n s in t h e urine. This finding was also s u p p o r t e d b y o u r previous d a t a t h a t liver h o m o g e n a t e s r e d u c e d n i t r o f u r a n to a m i n o f u r a n w i t h o u t p r o d u c i n g d e t e c t a b l e nitroso or h y d r o x y l a m i n e [ 1 8 ] . I n c i d e n t a l l y , a m i n o h e t e r o c y c l e s were n o t m u t a g e n i c [ 1 9 ] . T h e r e f o r e , t h e m u t a g e n i c activity o f the urine o f rats fed AF-2, F A N F T o r n i t r o f u r a n t o i n m a y be m a i n l y d u e t o t h e e x c r e t e d n i t r o f u r a n . I n d e e d , as s h o w n in Table IV, AF-2, A N F T and n i t r o f u r a n t o i n were det e c t e d i n ' t h e urine whereas t h e o t h e r n i t r o f u r a n s were not. F A N F T was def o r m y l a t e d b y a r y l f o r m a m i d a s e in several organs o f the rats [17] and exc r e t e d as A N F T in t h e urine. N o F A N F T was d e t e c t e d in the urine. A N F T was m u t a g e n i c in T A 1 0 0 [ 1 0 ] . T h e results d e m o n s t r a t e d t h a t 5-nitro-2furoic acid, 5 - n i t r o - 2 - f u r a m i d o x i m e and 5 - n i t r o f u r f u r y l i d e n e d i a c e t a t e w e r e m e t a b o l i z e d t o such an e x t e n t t h a t n o n e o f these c o m p o u n d s c o u l d be d e t e c t e d in the urine. F A N F T i n d u c e d u r i n a r y b l a d d e r c a n c e r in rats [19] and dogs [ 2 0 ] . T h e u r o t h e l i a l cells o f rat and dog c o u l d activate A N F T , the u r i n a r y m e t a b o l i t e o f F A N F T , and p r o d u c e m a c r o m o l e c u l e - b o u n d a d d u c t s ( u n p u b l i s h e d data). H a s h i m o t o and Kitagawa [21] f o u n d t h a t rat u r o t h e l i a l cells could be transf o r m e d b y N - b u t y l - N - ( 4 - h y d r o x y b u t y l ) n i t r o s a m i n e and N-butyl-N-(3-carb o x y p r o p y l ) n i t r o s a m i n e . T h e s e d a t a i n d i c a t e d t h a t chemical carcinogens
TABLE III EFFECT OF fl-GLUCURONIDASE ON THE MUTAGENIC ACTIVITY OF URINE OF RATS FED NITROFURANS Nitrofuran
Control AF-2 FANFT Nitrofurantoin 5-Nitro-2-furoic acid 5-Nitro-2-furamidoxime 5-Nitrofurfurylidene diacetate
Urine (pl/plate) a
2 2 0.2 2 0.2 20 20 20
Number of revertant colonies/plate (TA100) b Without enzyme
With enzyme c
96 170 508 92 602 108 165 138 95
82 168 516 102 715 125 165 144 139
a Urine was diluted in saline and 0.2 ml was added in each plate. b Average of duplicate determinations. c Each plate contained 5 mg of bacterial 13-glucuronidase.
74 TABLE IV DETECTION OF NITROFURANS IN THE URINE OF RATS Nitrofuran
R[ value on
Color b
Detected in urine
cellulose TLC a
AF-2 FANFT c ANFT c Nitrofurantoin 5-Nitro-2-fuvoic acid 5-Nitro-2-furamidoxime 5-Nitrofurfurylidene diacetate
0.33 0.14 0.23 0.55 0.88 0.63 0.45 d
Visible light
UV light
Y Y Y Y none Y none
DO Y DO Y D D D
Yes
No Yes Yes No No No
a Ascending chromatography with 2% formic acid solvent system. b y ; yellow; DO, dull orange; D, dark; none, invisible.
c Rat fed FANFT and only ANFT was detected in the urine. d Severe tailing on TLC. c o u l d be a c t i v a t e d in susceptible organs. T h e r e f o r e , c a r c i n o g e n i c i t y m a y be a f u n c t i o n o f t h e c o n c e n t r a t i o n o f t h e c a r c i n o g e n in t h e susceptible organ, and m a y explain w h y A F - 2 and F A N F T , w h i c h are m e t a b o l i z e d slowly, are carcinogens, w h e r e a s 5-nitro-2-furoic acid, 5 - n i t r o - 2 - f u r a m i d o x i m e and 5-nitrof u r f u r y l i d e n e d i a c e t a t e , w h i c h are m e t a b o l i z e d r a p i d l y , are n o n c a r c i n o g e n s . T h e failure of these n i t r o f u r a n s to i n d u c e t u m o r s m a y also be d u e to t h e i r w e a k m u t a g e n i c activity [1] or w e a k ability t o i n d u c e D N A d a m a g e [ 1 3 ] . N i t r o f u r a n t o i n has b e e n s h o w n to be n o n c a r c i n o g e n i c in rats [ 3 , 1 2 ] ; h o w ever, this c o m p o u n d has n o t b e e n t e s t e d u n d e r o p t i m a l c o n d i t i o n s [12] and t h e results h a v e b e e n c o n s i d e r e d inconclusive [ 1 1 ] . Since n i t r o f u r a n t o i n was m u t a g e n i c a n d was m e t a b o l i z e d at a slow rate, it m a y be a carcinogen. T h e urine o f m i c e fed F A N F T has b e e n s h o w n to be m u t a g e n i c to E. coli [ 2 2 ] . ACKNOWLEDGEMENTS We are grateful t o Dr. B.N. A m e s , U n i v e r s i t y o f California, B e r k e l e y , California, f o r p r o v i d i n g S. typhimurium strain T A 1 0 0 , Dr. H.S. R o s e n k r a n z , C o l u m b i a U n i v e r s i t y , N e w Y o r k , N e w Y o r k , f o r strain T A 1 0 0 F R 1 , and Dr. T. M a t s u s h i m a , U n i v e r s i t y o f T o k y o , T o k y o , J a p a n , f o r AF-2. We t h a n k Dr. G.T. B r y a n f o r m a n y h e l p f u l criticisms, and K. D e i g h t o n a n d D. S c o t t f o r t h e p r e p a r a t i o n of t h e m a n u s c r i p t . This s t u d y was s u p p o r t e d in p a r t b y U S P H S research grant C A 1 7 4 4 9 f r o m t h e N a t i o n a l C a n c e r I n s t i t u t e t h r o u g h t h e N a t i o n a l Bladder Cancer P r o g r a m . REFERENCES 1 D.R. McCalla and D. Voutsinos, On the mutagenicity of nitrofurans, Mutation Res., 26 (1974) 3.
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